Structure based and combinatorially selected oligonucleoside phosphorothioate and phosphorodithioate aptamer targeting AP-1 transcription factors

ABSTRACT

The present invention includes composition and methods for making and using a combinatorial library to identify modified thioaptamers that bind to, and affect the immune response of a host animal, transcription factors such as IL-6, NF-κB, AP-1 and the like. Composition and methods are also provided for the treatment of viral infections, as well as, vaccines and vaccine adjuvants are provided that modify host immune responses.

This work was supported by the following United States Government grantsDARPA (9624-107 FP), NIH (A127744) and NIEHS (ES06676). Without limitingthe scope of the invention, its background is described in connectionwith oligonucleotide agents and with methods for the isolation andgeneration thereof.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/472,890, filed May 23, 2003.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of thioaptamers,and more particularly, the use of thioaptamers for screening, includinghigh-throughput screening, of primary or secondary target molecules byusing thioated aptamers bound to a substrate with specific targeting tothe AP-1 family of transcription factors and for the treatment of viralinfections, as well as, vaccines and vaccine adjuvants that modify hostimmune responses.

BACKGROUND OF THE INVENTION

Virtually all organisms have nuclease enzymes that degrade rapidlyforeign DNA as an important in vivo defense mechanism. The use,therefore, of normal oligonucleotides as diagnostic or therapeuticagents in the presence of most bodily fluids or tissue samples isgenerally precluded. It has been shown, however, thatphosphoromonothioate or phosphorodithioate modifications of the DNAbackbone in oligonucleotides can impart both nuclease resistance andenhance the affinity for target molecules, such as for example thetranscriptional activating protein NF-κB.

Recent world events have heightened the awareness of possiblebioterrorist threats. Hemorrhagic fever viruses (category A bioweaponagents) have reportedly been weaponized by the former Soviet Union andthe United States (Borio et al., 2002; Hawley & Eitzen, 2001). Despitethe awareness of the potential of Viral Hemorrhagic Fever viruses(Lassa, Junin), Encephalitic viruses (West Nile, VEE) and other agentsboth as bioweapons and as emerging viral diseases, few therapeuticoptions are available to those infected. Apart from supportive therapy,the only drug for treating Arenavirus infections is Ribavirin and it isonly partially effective (McCormick et al, 1986a; Shulman, 1984; Enriaet al., 1987) while there are no efficacious drugs to treat victims ofWest Nile infections (Peterson and Marfin, 2002). There is an urgentneed to expand the current therapeutic armamentarium, which is hindered,at least in part, by a lack of in-depth knowledge concerning themechanisms of Arenaviral pathogenesis (Peters & Zaki, 2002).

Arenavirus pathogenesis stems from host immune response dysregulationand endothelial dysfunction (Peters & Zaki, 2002; Ignatyev et al., 2000;McCormick & Fisher-Hoch, 2002; Walker et al., 1982; McCormick et al.,1986b; Marta et al., 1999). West Nile pathogenesis is associated withthe inability of host immune response to limit virus replication tolevels below that required for viral invasion of the CNS (Solomon andVaughn, 2002).

Lassa fever, a human arenavirus hemorrhagic fever virus endemic in WestAfrica, affects up to 300,000 people annually and is responsible for upto 3000 deaths (McCormick, et al., 1987). Lassa Fever virus is difficultto study due to its hazardous nature (a BSL4 agent). Junin Virus is thecausative agent of Argentine hemorrhagic fever (AHF). The annualincidence varies between 100-4000 cases/yr. AHF has a case fatality rateof 15-30% and is also a BSL4 agent. A well-established animal model thatresembles Lassa Fever, using the non-pathogenic New World Arenavirus,Pichinde virus (Jahrling et al., 1981) has been used to study this classof pathogens. Serial passage of Pichinde virus in guinea pigs was usedto develop a virulent variant that produces a disease in guinea pigsthat mimics human Lassa Fever in many important respects including:viremia correlates with disease outcome (Johnson et al., 1987; Aronsonet al., 1994), a relative paucity of pathologic findings in lethallyinfected animals (Walker, et al., 1982; Connolly, et al., 1993),terminal vascular leak syndrome (Katz & Starr, 1990) and distribution ofviral antigens within the host (Connolly et al., 1993; Shieh et al.,1997; Aronson, unpublished data). Macrophage responses to the attenuatedPichinde virus, P2, with the virulent Pichinde variant, P18 as well asreassortants of the two variants (Zhang et al., 1999; Zhang et al.,2001; Fennewald et al., 2002) may be used to compare and modify theimmune response to viral infection.

West Nile virus (Category B virus) is a mosquito-borne flavivirus thatis a neuropathogen in humans, equines and avians (Solomon and Vaughn,2002; Petersen and Marfin, 2002). Humans become infected by the bite ofan infected mosquito. The viruses are then thought to replicate in theskin before being transported to the local lymph nodes. West Nile maythen spread via the blood to other organs including the liver, spleen,heart and kidney and eventually the brain. West Nile virus may spread tothe CNS via either hematogenous spread or via the olfactory mucosa wherethere is no blood-brain barrier. West Nile is an emerging pathogen inthe US, spreading across the country since it was first identified inNew York in 1999. As of Oct. 3, 2002, the CDC has reported 2530 cases ofWest Nile virus infection with 125 deaths in 32 states. West Nile isalso responsible for major outbreaks in other countries includingTunisia, Romania, Algeria, Russia and Israel among others. Case fatalityrates range from 4-29%. Age is a risk factor in the development ofsevere West Nile disease with many patients exhibiting substantialmorbidity. Presently, treatment for West Nile is limited to supportiveintervention. There is no evidence that either interferon or Ribavirintreatment is efficacious (Petersen and Marfin, 2002).

Arenavirus Hemorrhagic Fevers, such as Lassa fever, Junin, Argentinehemorrhagic fever, Bolivian hemorrhagic fever and Venezuelan hemorrhagicfever, have several features in common with sepsis and the systemicinflammatory response syndrome, including fulminant clinical course,fever, shock, capillary leak syndrome, decreased myocardialcontractility, abnormalities of coagulation and platelet function, andelevated serum levels of TNFα (Aronson et al., 1994; Cummins, 1990).Arenaviruses are non-cytopathic viruses with a tropism for macrophagesand other reticuloendothelial cells (Cummins, 1990; Peters et al.,1987); the pathogenesis of these diseases is believed to involveexcessive production of pro-inflammatory cytokines (Aronson et al.,1995; Peters et al., 1987). Unpublished data (Bausch et al., CDC) showcytokines to be massively activated in human Lassa fever, and alsoconfirm that Lassa virus can directly induce cytokine secretion byinfecting human macrophages in vitro (Mahanty et al., CDC, unpublished).Alternatively, there is evidence that a swift elaboration ofpro-inflammatory cytokines and early engagement of the (innate) immuneresponse may help protect of the infected host from lethal disease invarious hemorrhagic fever syndromes (Peters et al., 1987).

Endotoxic shock results from an innate, anaphylactic response tobacterial lipopolysaccharide (LPS). The NF-κB transcription factor, inconjunction with other cellular transcription factors, plays a criticalrole in gene activation, especially in acute phase and inflammatoryresponses (Baeuerele, 1998; Barnes and Karin, 1997), and in particularendotoxic shock, a complex pathophysiological state which is consideredto be an exaggerated or dysregulated systemic acute inflammatoryresponse syndrome initiated by the binding of bacterial LPS complexedwith lipopolysaccharide binding protein (LBP) to the CD14 receptor onmacrophages. A series of intracellular signaling events, in which NF-κBactivation figures importantly leads to enhanced transcription ofproinflammatory mediators, including TNFα, IL-1 and inducible nitricoxide synthase, ultimately promoting vasodilatation, capillaryleakiness, and myocardial suppression (Murphy et al., 1998). Inwell-established mouse endotoxemia models, rapid transient increases inNF-κB DNA-binding activity can be detected in the nuclei of macrophagesand other cell types (Boher, et al., 1997); similar observations havebeen made in human sepsis (Velasco et al., 1997).

The AP-1 transcription factor family include the dimeric basic regionleucine zipper proteins that belong to the Jun (c-Jun, JunB, JunD), Fos(c-Fos, FosB, Fra-1, Fra-2) Maf (c-Maf, MafB, MafA, MafG/F/K, Nrl) andATF/CREB (CREB, CREBP-2, ATF1, ATF2, LRF1/ATF3, ATF4, ATFa, ATF6, B-ATF,JDP1, JDP2) subfamilies which recognize either12-O-tetradecanoylphorbol-13-acetate (TPA) response elements(5′-TGAG/CTCA-3′) or cAMP response elements (CRE, 5′-TGACGTCA-3′)(Chinenov and Kerppola, 2001; Shaulian and Karin, 2002). Thesetranscription factor binding sites are elements in the promoters andenhancers of numerous mammalian genes including IL-2, IL-3, IL-4, IL-5,IFNβ, TNFα and GM-CSF (Chineov and Kerppola, 2001). The c-Jun protein isthe most potent transcription factor. The c-Fos proteins, which cannothomodimerize can form heterodimers with c-Jun and thereby enhance theirDNA binding activities. The c-Fos, and FosB proteins containtransactivation domains, however, Fra1, Fra2 and some splice variants ofFosB do not. CREB and ATF1 can form homodimers and heterodimers but donot combine with other ATF proteins. ATF2, ATFa, CREBP-2, ATF3, ATF4 andATF6 combine both with themselves and with specific Jun and/or Fosfamily members. C-Fos and Fra1 can heterodimerize with ATF4, but notwith ATF2 and ATF3.

There are numerous other possible homodimers and heterodimers possibleamong this large group of BZIP proteins. Jun, Fos and ATF family memberscan also bind to DNA upon association with certain Maf, C/EBP andnon-bZIP member factors like NF-κB, NFAT and Smad. This can direct AP-1components to promoter sequences that only slightly resemble consensusAP-1 and ATF motifs. This variation in dimer partner and DNA bindingsite specificity is assumed to provide AP1 subunits with a high level offlexibility in gene regulation. The regulation of AP-1 family oftranscription factor activity is complex but briefly regulation occursthrough: 1) changes in jun and fos gene transcription and mRNA turnover,2) Fos and Jun protein turnover, 3) post-translational modifications ofboth Fos, Jun other family proteins that modulate their activities, and4) interactions with other transcription factors (Shaulian and Karin,2001,2002). AP-1 activity is induced by growth factors, cytokines,neurotransmitters, polypeptide hormones, cell/matrix interactions,bacterial and viral infections and a variety of environmental stresses.These activators stimulate a series of signaling events that involve avariety of protein kinases including MAPKs, ERKs and JNKs. Members ofthe Fos and Jun protein families participate in the regulation of avariety of cellular processes including cell proliferation,differentiation, apoptosis, oncogenesis, inflammation, and immunity(Chinenov and Kerppola, 2001).

SUMMARY OF THE INVENTION

The present invention demonstrates the use of “thioaptamers™” to preventArenavirus and Flavivirus induced perturbations of the host responsethat lead to disease. Furthermore, the present invention provides fornovel therapeutic interventions for the treatment of hemorrhagic fevers,encephalitic viruses and other viral infections, resulting from theiruse as bioweapons or as emerging diseases. For example, modifiedthioaptamers were used to demonstrate modulation of NF-κB and AP-1 toincrease the survival of Arenavirus infected guinea pigs and miceinfected with West Nile virus in a well-established model system. Thepresent invention was also used to protect against viral infection witha neuropathologic viral infection. The modified thioaptamers of theinvention were created and used to protect mice challenged with WestNile virus in a well-established model system.

The present invention is based on the recognition that thiomodifiedaptamers may be designed, isolated and used to manipulate transcriptionfactors such as NFκB and AP-1 to interdict the pathogenetic sequence, oreven boost early protective innate immune responses (FIG. 1). Todemonstrate the feasibility of using the modified thioaptamers disclosedherein at physiological concentrations, animal model systems were usedthat models both severe fatal disease and self-limited infection withmild disease. For example, a well-recognized and widely used guinea pigmodel for Lassa Fever uses the New World arenavirus Pichinde (PIC)(Peters et al., 1987) was used and adapted to study pathogenesis bycomparing an attenuated variant of PIC (P2) and a closely relatedvirulent variant derived by serial guinea pig passage (P18) (Jahrling etal., 1981).

The present invention also uses the modified thio-aptamers to manipulateNF-κB levels in vivo. For example, the modified thioaptamers of thepresent invention were used to modify toxic shock via IκBαoverexpression increased mouse survival after high dose LPS challenge.The modified thioaptamers of the present invention may be used to targetthe five NF-κB/Rel family proteins, which combine to form 15 homo- andheterodimers. By targeting target one or more of the five NF-κB/Relfamily members, the present invention is used to modify one or more ofthe signaling pathways that regulate a specific signaling function upontranslocation across the cell nuclear membrane and binding to a gene'spromoter region.

While it is recognized that the AP-1 and NF-κB transcription factorfamilies both play key roles in the immune response and both representappropriate targets for therapies for viral infections, it has not beenpossible to modify in a physiologic manner their activities. The presentinvention allows for the modification of transcription factor activitiesusing modified thioaptamers that act under physiological conditions andat physiological levels to regulate transcriptional activation. Suchregulation may be used to modify responses to diseases involvingpathogenic or disfunctional inflammatory responses such as cancer, heartdisease, inflammatory bowel disease, rheumatoid arthritis and lupus.

The present invention was used to modulate induction of CREB, atranscription factor regulated by cyclic AMP (cAMP) signaling. Themodified thioaptamers were used to modulate CREB activity and weredemonstrated to modify virulent and attenuated Arenavirus infection. TheCREB protein is also a member of the AP-1 family of transcriptionfactors whose targeting by XBY-S2 has provided protection for animalsinfected with arenavirus and flavivirus. cAMP is a ubiquitous secondmessenger (Antoni et al., 2000) synthesized in cells by adenylylcyclases in response to many extra-cellular stimuli. Most cellulareffects of cAMP are mediated through the activation of cAMP dependentprotein kinases (PKA) (Sassone, 1995). PKA phosphorylation of substratesin all cellular compartments regulates a large array of cellularprocesses (Feliciello et al., 2001). Cyclic AMP induces changes in geneexpression that modulate macrophage apoptosis (von Knethen & Brüne,2000) and could contribute to pathogenic inflammatory conditions andsepsis. There is also evidence for functional cross talk between cAMPsignaling and the Jak/STAT pathway (Meloche et al., 2000). Agents thatincrease intracellular concentrations of cAMP inhibit IL-6 induced STATactivation in monocytes and interferon-β stimulated phosphorylation ofJak1, Tyk2, STAT1 and STAT2 in myeloma cells. Therefore, the modulationof the Jak/STAT pathway by cAMP is likely to play an important role inthe regulation of immune and inflammatory responses, which may beregulated using the modified thiopatamers of the present invention.

The present invention provides a number of advantages due to the use ofmodified thioaptamers and combinatorial selection methods. The presentinvention provides very high affinity-nM to sum-nM (≧monoclonal IgMsand >non-substituted aptamers), target-specific aptamers, demonstratingsingle protein target binding within cellular extracts. The modifiedthioaptamers have greater resistance to cellular or serum nucleasedegradation than normal backbone aptamers, or proteases towardsantibodies. Due to the increased nuclease resistance, the aptamersdisclosed herein may be packaged to have indefinite shelf-life, ease ofstorage as lyophilized powders at room temperatures, unlike unmodifiedRNA or antibodies and are relatively inexpensive to produce.Furthermore, the methods and compositions disclosed herein allow forhigh reproducibility in quality control, unlike diasteromeric mixturesfor non-stereospecifically produced monothiophosphate aptamers, orprotein production of antibodies. Finally, the use of bead-basedthioaptamer libraries or library of libraries provides largecombinatorial libraries readily selected by multicolor flow cytometry atvery high speeds (10⁸/hr).

In one embodiment, the present invention is a system and method foridentifying both thioaptamer sequences and binding one or more proteinsthat include the steps of, incubating a thioaptamer library with asample suspected of including one or more proteins, e.g., targetproteins. The proteins that bind the thioaptamers are selected from theone or more thioaptamers of the library to which protein has bound, theproteins are identified using mass spectrometry and/or the thioaptameris sequenced using, e.g., a method that includes PCR amplifying theaptamer followed by, cloning and sequencing. The thioaptamers may be onbeads, e.g., as part of a one-bead, one-thioaptamer library and may besequenced, e.g., directly on the beads.

The system and method may also include the step of separating theprotein into fragments prior to separation by liquid chromatographyfollowed by mass spectrometry. In an alternative method, the step ofidentifying the protein by mass spectrometry (MS) may be, e.g.,time-of-flight (TOF) MS. In one example, prior to the step ofidentifying the protein, the protein may be extracted and then separatedby liquid chromatography. The identification of the protein may be bysurface enhanced laser desorption ionization (SELDI) or matrix assistedlaser desorption ionization (MALDI) prior to MS. The thioaptamers may beattached to beads or a substrate, e.g., a semiconductor substrate.Semiconductor substrates may be used as arrays that permit detection ofprotein:thioaptamer binding and may further include detectors that areintegral with the substrate (e.g., capacitance coupled devices) or evensurface metal for surface plasmon resonance (SPR) detection. Thethioaptamer library may even be a microarray on a substrate that doesnot include an integral detected, e.g., a glass slide on which athioaptamer library has been disposed using, e.g., photolithography ordigital optical chemistry. The location of protein binding on such amicroarray may be detected using well known protein detection methods,e.g., fluorescence. The protein for use with the invention may beprotein from a crude extract or even partially purified or isolated,e.g., one or more proteins isolated from a gel.

The system and method disclosed herein may further include the use ofbinding the thioaptamers to beads and sorting the beads to isolate andidentify proteins that have specifically bound to the thioaptamers. Forexample, when using a thioaptamer library of beads, the beads may besorted based on protein binding, e.g., based on fluorescence labeling ofthe aptamer and/or the protein using a flow-cytometer. The protein maybe from a cell extract, which may even be a cell extract from a virallyinfected or diseased cell. Generally, the thioaptamers are attached tobeads and the beads are substantially protein-free. When using aone-bead, one-thioaptamer (ODN) library or even a library of librariesthe thioaptamers may be one or more beads that include an [S]-ODN and/or[S₂]-ODN combinatorial libraries. The ODNs may be single or doublestranded and may include thio-modifications to one or both of thestrands

In one embodiment of the present invention the thioaptamer libraryincludes, or is designed to include, sequence motifs for high affinitywith cellular proteins selected from proteins that are members of, e.g.,the AP-1, RBP-Jκ, NF-κB, NF IL-6, CREB and GRE protein families, andcombinations thereof. In operation, the system and method may alsoinclude the step of comparing a first and a second incubation of one ormore beads to a first and a second sample, respectively, whereindifferences in binding are used to detect proteins that expresseddifferentially, e.g., proteins from a virally-infected (or diseased)cell or even a cancer cell. In an alternative embodiment, the method mayalso include the steps of binding the one or more thioaptamers to one ormore beads, incubating the one or more thioaptamer beads with a cellextract from a cell wherein proteins from the cell extract are labeledwith a first dye; incubating the one or more thioaptamers beads with acell extract from a diseased-cell wherein proteins from thediseased-cell extract are labeled with a second dye, incubating the oneor more thioaptamers beads with a cell extract from a diseased-cellpre-treated with thioaptamers or other drugs, wherein the proteins ofthe diseased-cell but drug-treated, are labeled with a third dye; andperforming a three-color flow cytometry that measured the relativelevels of the first, second and third dyes.

Another embodiment of the present invention is a complex combinatoriallibrary that includes one or more concatenated thio-modified aptamers,wherein at least a portion of each of the aptamers is partiallythio-modified. The one or more concatenated thioaptamers may be bound toa substrate, e.g., one or more beads, a semiconductor, a surface plasmonresonance surface (e.g., gold), a multi-well plate and the like. Theconcatenated aptamer may include two or more concatenated thio-modifiedaptamers, wherein one or more of the aptamers is partiallythio-modified. In one example, the two or more concatenated thioaptamersmay include nucleic acid sequences suspected of binding to nuclearregulatory factors, and may even be a library of thioaptamers. Moreparticularly, the two or more concatenated thioaptamers may includenucleic acid sequences suspected of binding: NF-κB, RBP-Jκ, AP-1, NFIL-6, SP-1, GRE, SRE and the like. In one example, one or more of thethioaptamers may be a library of aptamers that binds to one or moretranscription factors and includes sequences or sequence motifs fortranscription factor binding, e.g., a NF-κB, a RBP-Jκ, an AP-1, an NFIL-6, an SP-1, a GRE, an SRE motif and/or mixtures thereof.

The complex combinatorial library made by a method that includes thesteps of synthesizing an aptamer bead library having a first thioaptamerand concatenating to each of the first thioaptamers a second aptamer orthioaptamer suspected of binding to, e.g., a nuclear regulatory factor.In fact, the first and second thioaptamers may even be suspected ofbinding the same nuclear regulatory factor or a different nuclearregulatory factor. Yet another embodiment of the present invention is amethod of identifying a thio-modified therapeutic agent that includesmixing a sample suspected of including a DNA binding protein with aconcatenated first and second thioaptamer under binding conditions andisolating the one or more DNA binding proteins that bind specifically tothe concatenated aptamers.

Another embodiment of the present invention is a composition, adjuvant,vaccine and method of modifying an immune response that includesproviding a host cell with aptamers that suppress the activity of anuclear regulatory factor critical for activation of an immune response.The immune response may be an innate immune response, a cytotoxic or ahelper T cell immune response. In one embodiment the thioaptamermodified the immune response by shifting the helper 1-type (Th1) to Thelper 2-type (Th2) ratio. The immune response that is modified may beto a virus, a bacteria, a fungus, a cancer, a self-antigen, aheterologous antigen, a retrovirus, a hemorraghic virus or aneuropathologic virus, e.g., West Nile Virus. The immune response thatis modified may be modified in vivo, in vitro and/or ex vivo. Themodification of the immune response may be an increase or decrease ofthe immune response as measured by, e.g., antibody production, cytotoxicT cell activation, cytokine release, apoptosis, cell proliferation, cellkilling, chromium release, nucleic or amino acid uptake or release andother methods known to those skilled in the immunological arts.

In one specific embodiment, the type of helper T cell response may bemodified by providing a host or target cell with one or morethioaptamers that suppress the activity of a nuclear regulatory factorcritical for activation of, e.g., a helper T cell response. The T cellimmune response may be to, e.g., a virus, a bacteria, a fungus, acancer, a self-antigen, a heterologous antigen, a retrovirus, ahemorraghic virus or even a neuropathologic virus. The modification tothe immune response may be to a challenge to the innate or the adaptiveimmune response. The helper T cell response may be a T helper 1-typeresponse or a T helper 2-type response.

Another embodiment of the invention is a vaccine that includes anantigen and a thioaptamer. The vaccine may be to an antigen from, e.g.,a virus, a bacteria, a fungus, a cancer, a self-antigen, a heterologousantigen, a xenoantigen, a retrovirus, a hemorraghic virus or aneuropathologic virus. The vaccine may be provided in a lyophilized, aparticulate or even a dissolved form and may even include one or morepharmaceutically acceptable salts, diluents, preservatives and the like.The antigen may be, e.g., a live-attenuated antigen or aheat-inactivated antigen. Examples of viral antigens include:hemorrhagic fever viruses, which include viruses from different viralfamilies, e.g., Ebola, Marburg, Lassa fever, New World Arenavirus, RiftValley Fever, yellow fever, Omsk hemorrhagic fever and Kyasanur ForestDisease viruses. Four viral families are generally implicated inhemorrhagic fever infections, including: (1) Arenaviridae (Lassa, Junin,Machupo, Guanarito, and Sabia viruses, which are the causative agents ofLassa fever and Argentine, Bolivian, Venezuelan, and Brazilianhemorrhagic fevers, respectively); (2) Filoviridae (Ebola and Marburg);(3) Flaviviridae (yellow fever, Omsk hemorrhagic fever, and KyasanurForest disease viruses); (4) Bunyaviridae (Rift Valley fever (RFV),Congo-Crimean hemorrhagic fever. Another target viral family includesHantaviruses. Another antigen for targeting includes neuropathologicviruses, e.g., St. Louis encephalitis, Western equine encephalitis,Eastern equine encephalitis, California encephalitis serogroup (e.g.,LaCrosse, Jamestown Canyon, Snowshoe Hare, Trivittatus, Keystone, andCalifornia encephalitis viruses), Powassan encephalitis, Venezuelanequine virus, Argentine equine encephalitis virus, Cache Valley virusand West Nile virus. Neuropathologic viruses fall into various viralfamilies and are characterized by symptoms that include: fever ofvariable severity associated with neurologic symptoms ranging fromheadache to aseptic meningitis or encephalitis, headache, confusion orother alteration of the senses, nausea and vomiting. Signs may includefever, meningismus, cranial nerve palsies, paresis or paralysis, sensorydeficits, altered reflexes, convulsions, abnormal movements and coma ofvarying degree.

The thioaptamers of the present invention may be an adjuvant that formspart of a vaccine, such as a composition that includes one or morepartially thio-modified or even concatenated aptamers that modulate animmune response. When used as a vaccine, that thioaptamer adjuvant mayalso include at least one antigen. In addition to the exampleshereinabove, the antigen may be a pathogen-associated molecular patternantigen, e.g., a CpG molecule, a saccharide, a lectin, a polysaccharideand the like. As with the thioaptamers described hereinabove theadjuvant thioaptamer may include sequences for specific recognition andbinding to nuclear regulatory factors, e.g., NF-AT, NF-κB, RBP-Jκ, AP-1,NF IL-6, SP-1, GRE and SRE. Examples of partially thioaptamers includeone or more of the aptamers of SEQ ID NOS.: 2, 3, 4, 5, 6, 7, 8 and 9.

The thioaptamer may an adjuvant that includes one or more partiallythioaptamers that bind to, e.g., a DNA binding protein and modulate animmune response, e.g., an innate or an adaptive immune response. Theadjuvant may be provided with a physiologically acceptable aqueousvehicle, in a lyophilized, a particulate or even a dissolved form withor without an antigen, e.g., the antigen described hereinabove. Thethioaptamer may be specific for one or more downstream nuclearregulatory factors that transduce a intracellular signal from aToll-Like receptor, e.g., a Toll-Like receptor 2, a Toll-Like receptor 4or a pathogen-associated molecular pattern receptor. The adjuvant may bea partially thioaptamer selected from SEQ ID NOS.: 2, 3, 4, 5, 6, 7, 8,9, 56 and/or 58. Another embodiment of the present invention is a T celladjuvant that includes, e.g., a peptide antigen and an aptamer whereinat least a portion of at least one nucleotide in the thioaptamer isthiophosphate-modified.

The present invention also includes a method of treating a hemorraghicviral infection that includes the steps of identifying a patientsuspected of being infected with a hemorraghic virus and providing thepatient with a therapeutic amount of a thioaptamer specific for atranscription factor involved in viral propagation or the immune cellresponse related to the virus. The transcription factor may be, e.g.,NF-κB, RBP-Jκ, AP-1, NF IL-6, SP-1, GRE, SRE, mixtures thereof and thelike. The thioaptamer will generally bind specifically to a protein,e.g., a transcription factor and may also include one or more of theaptamers of SEQ ID NOS.: 2, 3, 4, 5, 6, 7, 8 and 9, e.g.,

XBY-6: 5′-CCAGGAGAT_(S2)T_(S2)CCAC-3′ SEQ ID NO.: 13′-GG_(S2)TCC_(S2)TC_(S2)TAAGG_(S2)TG-5′ XBY-S2:5′-CCAGT_(S2)GACT_(S2)CAGT_(S2)G-3′ SEQ ID NO.: 23′-GG_(S2)TCAC_(S2)TGAG_(S2)TCAC-5′ XBY-S1:5′-T_(S2)T_(S2)GCGCGCAACAT_(S2)G-3′ SEQ ID NO.: 33′-AACGCGCG_(S2)T_(S2)TG_(S2)TAC-5′ XBY-C2: 5′-CCAGTGACTCAGTG-3′ SEQ IDNO.: 4 3′-GGTCACTGAGTCAC-5′ XBY-C1: 5′-TTGCGCGCAACATG-3′ SEQ ID NO.: 53′-AACGCGCGTTGTAC-5′ 5′-tGTGcAGGGACTgAtGaCGGt-3′, SEQ ID NO.: 65′-CtGTGCatCGAaGTTtGCAtTt-3′, SEQ ID NO.: 75′-AtGcAcAtCtCaGgAtGaCGGt-3′, SEQ ID NO.: 85′-AGTTGcAGGtCaGgACCCAtTt-3′, SEQ ID NO.: 9wherein the lowercase letters represent the thiophosphate 3′ to thebase. In one examples, the method of treatment may be directed to aneuropathologic viral infection and include the steps of identifying apatient suspected of being infected with a neuropathologic virus; andproviding the patient with a therapeutic amount of a partiallythioaptamer specific for transcription factor involved in immune cellactivation. A thioaptamer for use in the method of treatment may beXBY-S2.

Yet another embodiment of the present invention is a method formodifying an immune response that includes administering a compositionthat includes an antigen and one or more partially thio-modifiedaptamers or thioaptamers. The modifications to the immune responseinclude, e.g., activation or deactivation of the innate immune responseand/or modifications to the type of immune response mounted (humoralversus cell-based) such as a change in the profile of helper T cellinvolved with or “lead” the immune response. The composition may alsoinclude cytokines, e.g., interleukin-1 (IL-1), interleukin-2 (IL-2),interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5),interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8),interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12),interleukin-13 (IL-13), Type I Interferon, Type II Interferon, tumornecrosis factor alpha (TNF-alpha), transforming growth factor-beta(TGF-beta), lymphotoxin migration inhibition factor,granulocyte-macrophage colony-stimulating factor (GM-CSF),monocyte-macrophage CSF, granulocyte CSF, vascular epithelial growthfactor (VEGF), angiogenin, transforming growth factor (TGF-alpha),fibroblast growth factor, angiostatin, endostatin, mixtures orcombinations thereof. The composition may also include one or moreantigens, e.g., lipid A, phospholipase A2, endotoxins, staphylococcalenterotoxin B, heat shock proteins (HSPs), carbohydrates, Rh factors,DNA, nucleotides, RNA, mRNA, MART, MAGE, BAGE, GAGE, DAGE, mutant p53,tyrosinase, or a combination thereof. The aptamer may stimulatespecialized antigen presenting cells (APCs), e.g., macrophages,dendritic cells and B cells or non-specialized immune or even non-immunecells. The aptamer may activate an innate immune response, e.g., throughToll-Like receptors that stimulate lymphocytes such as APCs, B cells andT cells. In one example, the aptamer activates an innate immune responsethat includes the simultaneous activation of macrophages and dendriticcells and of B cells and T cells. The aptamer may stimulate or suppressthe immune response.

In one specific embodiment, the present invention includes a method forenhancing vaccine efficacy by administering a composition that includesa partially thioaptamer specific for a DNA binding protein and anantigen to a subject animal. The aptamer may also include a carriermolecule, e.g., liposomes, microcapsules, microspheres, mixtures orcombinations thereof. The target immune response may be, e.g., to acancer or a pathogenic infection. Alternatively, the target immuneresponse may be an anaphylactic shock, allergic rhinitis, eczema,urticaria, anaphylaxis, transplant rejection, systemic lupuserthymatosus, rheumatoid arthritis, seronegative spondyloarthritides,Sjogren's syndrome, systemic sclerosis, polymyositis, dermatomyositis,Type I Diabetes Mellitus, Acquired Immune Deficiency Syndrome,Hashimoto's thyroiditis, Graves' disease, Addison's disease,polyendocrine autoimmune disease, hepatitis, sclerosing cholangitis,primary biliary cirrhosis, pernicious anemia, coeliac disease,antibody-mediated nephritis, glomerulonephritis, Wegener'sgranulomatosis, microscopic polyarteritis, polyarteritis nodosa,pemphigus, dermatitis herpetiformis, psoriasis, vitiligo, multiplesclerosis, encephalomyelitis, Guillain-Barre syndrome, MyastheniaGravis, Lambert-Eaton syndrome, sclera, episclera, uveitis, chronicmucocutaneous candidiasis, Bruton's syndrome, transienthypogammaglobulinemia of infancy, myeloma, X-linked hyper IgM syndrome,Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune hemolyticanemia, autoimmune thrombocytopenia, autoimmune neutropenia,Waldenstrom's macroglobulinemia, amyloidosis, chronic lymphocyticleukemia, or non-Hodgkin's lymphoma. The partially thioaptamer may bespecific for a DNA binding protein, a cellular protein, a cell surfaceprotein, a saccharide or lipid or combinations thereof. When provided invaccine form, the thioaptamer (thioaptamer) and an antigen may beprovided in dry form or even be disposed in a vehicle suitable for oral,intramuscular, subcutaneous, intravenous or parenteral administration,e.g., in a sterile saline solution. The partially thioaptamer may bespecific for AP-1, NF-κB, NF IL-6, or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 is a schematic representation for immune responses postinfection, in the left panel, Target A represents immune responseclearing virus with patient survival, in the right panel Target Brepresents cytopathogenic immune response resulting in shock;

FIG. 2 is a graph that shows the production of TNF-α in P388D1 cells.Cells were treated with polyI/C (25 μg/ml) and media samples were takenat indicated times, the TNF-α levels in the media were determined usingcommercially available ELISA;

FIGS. 3A, 3B and 3C are bar graphs that show the production of by P388cells infected with P2 or P18 taken three days post-infection andassayed for TNF-α (3A), IL-6 (3B) and IL-12 (3C);

FIG. 4 is a gel that shows that the XBY-S2 aptamer binds specifically toproteins in 70Z/3 cell nuclear extracts and recombinant human AP-1;

FIG. 5 is a gel that shows a supershift analysis using a variety ofantibodies specific for various members of the AP-1 transcription factorfamily;

FIG. 6 is a gel with a comparison of XBY-6 and Igκ oligonucleotidebinding to proteins in 70Z/3 cell nuclear extracts in which multipleNF-κB dimers are shown to bind the Igκ oligonucleotide, with specificbinding of only p50 (or p 105) containing dimers to XBY-6;

FIG. 7 is a gel that shows that XBY-S2 eliminates AP1 DNA bindingactivities in macrophages treated with liposomes with and without theindicated aptamers for 24 hours, wherein the nuclear extracts wereanalyzed by electrophoretic mobility shift assay (EMSA) with the AP-1and NF-κB oligonucleotide probes;

FIG. 8 is a graph that shows the secretion of TNFα as measured by ELISAof Mouse P388D1 macrophage cultures were treated with XBY-S2 for 12hours followed by stimulation with PolyI/C and harvested at 24 hrs;

FIG. 9 is a graph of IL-6 production assayed by ELISA of mouse P388D1macrophage cultures treated with XBY-S2 for 12 hours followed bystimulation with PolyI/C and harvested at 24 hrs;

FIG. 10 is a graph that shows survival curves following Pichinde P18infection in guinea pigs treated with the NF-κB aptamer, XBY-6, thescrambled control, B92, or vehicle, MT, of animals infected by injectionof 1000 pfu of Pichinde P18 at day 0, treatment consisted ofintraperitoneal injections at days 0, 1 and 2;

FIG. 11 is a graph that shows survival curves of guinea pigs withthioaptamers for infection by arenavirus;

FIG. 12 is a graph that shows survival curves following West Nile Virusinfection in guinea pigs treated with the NF-κB aptamer XBY-6, the AP-1aptamer XBY-S2, or the liposome vehicle of animals infected by injectionwith lethal doses of West Nile Virus;

FIG. 13 are graphs that show SELDI detection of recombinant p50 usingEpoxy-activated ProteinChip Arrays with XBY-6 (top), IgκB 22-mer duplex(middle) or control, poly(dI.dC) (bottom) covalently linked to surfaces;

FIG. 14 are graphs that show the detection of recombinant p50 on gelbeads using XBY-6. Top two SELDI MS extract from beads spotted onto NP20ProteinChip. Bottom two SELDI spectra taken on beads themselves, inwhich the control is no XBY-6 covalently attached to beads withaminolinker; and

FIG. 15 is a graph that shows the SELDI MS capture of endogenous p50(p105) from nuclear extracts on Ciphergen PS20 Proteinchip Arrays, thetopgraph shows covalently linked XBY-6 to array surface, in the bottom,control no XBY-6 linked to surface.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As used herein, “synthesizing” of a random combinatorial library refersto chemical methods known in the art of generating a desired sequence ofnucleotides including where the desired sequence is random. Typically inthe art, such sequences are produced in automated DNA synthesizersprogrammed to the desired sequence. Such programming can includecombinations of defined sequences and random nucleotides.

“Random combinatorial oligonucleotide library” means a large number ofoligonucleotides of different sequence where the insertion of a givenbase at given place in the sequence is random. “PCR primer nucleotidesequence” refers to a defined sequence of nucleotides forming anoligonucleotide which is used to anneal to a homologous or closelyrelated sequence in order form the double strand required to initiateelongation using a polymerase enzyme. “Amplifying” means duplicating asequence one or more times. Relative to a library, amplifying refers toen masse duplication of at least a majority of individual members of thelibrary.

As used herein, “thiophosphate” or “phosphorothioate” are usedinterchangeably to refer analogues of DNA or RNA having sulphur in placeof one or more of the non bridging oxygens bound to the phosphorus.Monothiophosphates or phosphoromonothioates [αS] have only one sulfurand are thus chiral around the phosphorus center. Ditbiophosphates aresubstituted at both oxygens and are thus achiral. Phosphoromonothioatenucleotides are commercially available or can be synthesized by severaldifferent methods known in the art. Chemistry for synthesis of thephosphorodithioates has been developed by one of the present inventorsas set forth in U.S. Pat. No. 5,218,088 (issued to Gorenstein, D. G. andFarschtschi, N., Jun. 8, 1993 for a Process for PreparingDithiophosphate Oligonucleotide Analogs via NucleosideThiophosphoramidite Intermediates), relevant portions incorporatedherein by reference.

As used herein, the terms “thio-modified aptamer” and “thioaptamer” areused interchangeably to describe oligonucleotides (ODNs) (or librariesof thioaptamers) in which one or more of the four constituent nucleotidebases of an oligonucleotide are analogues or esters of nucleotides thatnormally form the DNA or RNA backbones and wherein such modificationconfers increased nuclease resistance. For example, the modifiednucleotide aptamer can include one or more phosphorothioate orphosphordithioate linkages selected from dATP(αS), dTTP(αS), dCTP(αS)and dGTP(αS), dATP(αS₂), dTTP(αS₂), dCTP(αS₂) and dGTP(αS₂). In anotherexample, no more than three adjacent phosphate sites of the modifiednucleotide aptamer are replaced with phosphorothioate groups. In yetanother example, at least a portion of non-adjacent dA, dC, dG, or dTphosphate sites of the modified nucleotide aptamer are replaced withphosphorothioate groups. In another example of a thioaptamer, all of thenon-adjacent dA, dC, dG, or dT phosphate sites of the modifiednucleotide aptamer are replaced with phosphorothioate groups; all of thenon-adjacent dA, dC, dG, and dT phosphate sites of the modifiednucleotide aptamer are replaced with phosphorothioate groups; orsubstantially all non-adjacent phosphate sites of the modifiednucleotide aptamer are replaced with phosphorothioate groups. In stillanother embodiment of the present invention, no more than three adjacentphosphate sites of the modified nucleotide aptamer are replaced withphosphorodithioate groups. The thioaptamers may be obtained by addingbases enzymatically using a mix of four nucleotides, wherein one or moreof the nucleotides is a mix of unmodified and thiophosphate-modifiednucleotides, to form a partially thiophosphate-modified thioaptamerlibrary. In another example of “thioaptamers” these are made by addingbases to an oligonucleotide wherein a portion of the phosphate groupsare thiophosphate-modified nucleotides, and where no more than three ofthe four different nucleotides are substituted on the 5′-phosphatepositions by 5′-thiophosphates in each synthesized oligonucleotide arethiophosphate-modified nucleotides.

Thiophosphate nucleotides are an example of modified nucleotides.“Phosphodiester oligonucleotide” means a chemically normal (unmodified)RNA or DNA oligonucleotide. Amplifying “enzymatically” refers toduplication of the oligonucleotide using a nucleotide polymerase enzymesuch as DNA or RNA polymerase. Where amplification employs repetitivecycles of duplication such as using the “polymerase chain reaction”, thepolymerase may be, e.g., a heat stable polymerase, e.g., of Thermusaquaticus or other such polymerases, whether heat stable or not.

“Contacting” in the context of target selection means incubating aoligonucleotide library with target molecules. “Target molecule” meansany molecule to which specific aptamer selection is desired.“Essentially homologous” means containing at least either the identifiedsequence or the identified sequence with one nucleotide substitution.“Isolating” in the context of target selection means separation ofoligonucleotide/target complexes, preferably DNA/protein complexes,under conditions in which weak binding oligonucleotides are eliminated.

By “split synthesis” it is meant that each unique member of thecombinatorial library is attached to a separate support bead on a two(or more) column DNA synthesizer, a different thiophosphoramidite orphosphoramidite is first added onto both identical supports (at theappropriate sequence position) on each column. After the normal cycle ofoxidation (or sulfurization) and blocking (which introduces thephosphate, monothiophosphate or dithiophosphate linkage at thisposition), the support beads are removed from the columns, mixedtogether and the mixture reintroduced into both columns. Synthesis mayproceed with further iterations of mixing or with distinct nucleotideaddition.

Aptamers may be defined as nucleic acid molecules that have beenselected from random or unmodified oligonucleotides (“ODN”) libraries bytheir ability to bind to specific targets or “ligands.” An iterativeprocess of in vitro selection may be used to enrich the library forspecies with high affinity to the target. The iterative process involvesrepetitive cycles of incubation of the library with a desired target,separation of free oligonucleotides from those bound to the target andamplification of the bound ODN subset using the polymerase chainreaction (“PCR”). The penultimate result is a sub-population ofsequences having high affinity for the target. The sub-population maythen be subcloned to sample and preserve the selected DNA sequences.These “lead compounds” are studied in further detail to elucidate themechanism of interaction with the target.

Dosage forms. A dosage unit for use of the aptamers and partiallythioaptamers of the present invention, may be a single compound ormixtures thereof with other compounds, e.g., a potentiator. Thecompounds may be mixed together, form ionic or even covalent bonds. Theaptamers and partially thioaptamers of the present invention may beadministered in oral, intravenous (bolus or infusion), intraperitoneal,subcutaneous, or intramuscular form, all using dosage forms well knownto those of ordinary skill in the pharmaceutical arts. Depending on theparticular location or method of delivery, different dosage forms, e.g.,tablets, capsules, pills, powders, granules, elixirs, tinctures,suspensions, syrups, and emulsions may be used to provide the aptamersand partially thioaptamers of the present invention to a patient in needof therapy that includes the aptamers and partially thioaptamers. Theaptamers and partially thioaptamers may also be administered as any oneof known salt forms.

Aptamers and partially thioaptamers is typically administered inadmixture with suitable pharmaceutical salts, buffers, diluents,extenders, excipients and/or carriers (collectively referred to hereinas a pharmaceutically acceptable carrier or carrier materials) selectedbased on the intended form of administration and as consistent withconventional pharmaceutical practices. Depending on the best locationfor administration, the aptamers and partially thioaptamers may beformulated to provide, e.g., maximum and/or consistent dosing for theparticular form for oral, rectal, topical, intravenous injection orparenteral administration. While the aptamers and partially thioaptamersmay be administered alone, it will generally be provided in a stablesalt form mixed with a pharmaceutically acceptable carrier. The carriermay be solid or liquid, depending on the type and/or location ofadministration selected.

Techniques and compositions for making useful dosage forms using thepresent invention are described in one or more of the followingreferences: Ansel, Introduction to Pharmaceutical Dosage Forms 2ndEdition (1976); Remington's Pharmaceutical Sciences, 17th ed. (MackPublishing Company, Easton, Pa., 1985); Advances in PharmaceuticalSciences (David Ganderton, Trevor Jones, Eds., 1992); Advances inPharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, JamesMcGinity, Eds., 1995); Aqueous Polymeric Coatings for PharmaceuticalDosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (JamesMcGinity, Ed., 1989); Pharmaceutical Particulate Carriers: TherapeuticApplications: Drugs and the Pharmaceutical Sciences, Vol 61 (AlainRolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (EllisHorwood Books in the Biological Sciences. Series in PharmaceuticalTechnology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); ModernPharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S.Banker, Christopher T. Rhodes, Eds.), and the like, relevant portionsincorporated herein by reference.

For example, the aptamers and partially thioaptamers may be included ina tablet. Tablets may contain, e.g., suitable binders, lubricants,disintegrating agents, coloring agents, flavoring agents, flow-inducingagents and/or melting agents. For example, oral administration may be ina dosage unit form of a tablet, gelcap, caplet or capsule, the activedrug component being combined with an non-toxic, pharmaceuticallyacceptable, inert carrier such as lactose, gelatin, agar, starch,sucrose, glucose, methyl cellulose, magnesium stearate, dicalciumphosphate, calcium sulfate, mannitol, sorbitol, mixtures thereof, andthe like. Suitable binders for use with the present invention include:starch, gelatin, natural sugars (e.g., glucose or beta-lactose), cornsweeteners, natural and synthetic gums (e.g., acacia, tragacanth orsodium alginate), carboxymethylcellulose, polyethylene glycol, waxes,and the like. Lubricants for use with the invention may include: sodiumoleate, sodium stearate, magnesium stearate, sodium benzoate, sodiumacetate, sodium chloride, mixtures thereof, and the like. Disintegratorsmay include: starch, methyl cellulose, agar, bentonite, xanthan gum,mixtures thereof, and the like.

The aptamers and partially thioaptamers may be administered in the formof liposome delivery systems, e.g., small unilamellar vesicles, largeunilamallar vesicles, and multilamellar vesicles, whether charged oruncharged. Liposomes may include one or more: phospholipids (e.g.,cholesterol), stearylamine and/or phosphatidylcholines, mixturesthereof, and the like.

The aptamers and partially thioaptamers may also be coupled to one ormore soluble, biodegradable, bioacceptable polymers as drug carriers oras a prodrug. Such polymers may include: polyvinylpyrrolidone, pyrancopolymer, polyhydroxylpropylmethacrylamide-phenol,polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues, mixtures thereof, and the like.Furthermore, the aptamers and partially thioaptamers may be coupled oneor more biodegradable polymers to achieve controlled release of theaptamers and partially thioaptamers, biodegradable polymers for use withthe present invention include: polylactic acid, polyglycolic acid,copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacylates, and crosslinked or amphipathicblock copolymers of hydrogels, mixtures thereof, and the like.

In one embodiment, gelatin capsules (gelcaps) may include the aptamersand partially thioaptamers and powdered carriers, such as lactose,starch, cellulose derivatives, magnesium stearate, stearic acid, and thelike. Like diluents may be used to make compressed tablets. Both tabletsand capsules may be manufactured as immediate-release, mixed-release orsustained-release formulations to provide for a range of release ofmedication over a period of minutes to hours. Compressed tablets may besugar coated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere. An enteric coating may be used to provideselective disintegration in, e.g., the gastrointestinal tract.

For oral administration in a liquid dosage form, the oral drugcomponents may be combined with any oral, non-toxic, pharmaceuticallyacceptable inert carrier such as ethanol, glycerol, water, and the like.Examples of suitable liquid dosage forms include solutions orsuspensions in water, pharmaceutically acceptable fats and oils,alcohols or other organic solvents, including esters, emulsions, syrupsor elixirs, suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Such liquid dosage forms may contain, forexample, suitable solvents, preservatives, emulsifying agents,suspending agents, diluents, sweeteners, thickeners, and melting agents,mixtures thereof, and the like.

Liquid dosage forms for oral administration may also include coloringand flavoring agents that increase patient acceptance and thereforecompliance with a dosing regimen. In general, water, a suitable oil,saline, aqueous dextrose (e.g., glucose, lactose and related sugarsolutions) and glycols (e.g., propylene glycol or polyethylene glycols)may be used as suitable carriers for parenteral solutions or even fordelivery via a suppository. Solutions for parenteral administrationinclude generally, a water soluble salt of the active ingredient,suitable stabilizing agents, and if necessary, buffering salts.Antioxidizing agents such as sodium bisulfite, sodium sulfite and/orascorbic acid, either alone or in combination, are suitable stabilizingagents. Citric acid and its salts and sodium EDTA may also be includedto increase stability. In addition, parenteral solutions may includepharmaceutically acceptable preservatives, e.g., benzalkonium chloride,methyl- or propyl-paraben, and/or chlorobutanol. Suitable pharmaceuticalcarriers are described in Remington's Pharmaceutical Sciences, MackPublishing Company, a standard reference text in this field, relevantportions incorporated herein by reference.

Intranasal and Nasal. For direct delivery to the nasal passages,sinuses, mouth, throat, esophagous, tachea, lungs and alveoli, theaptamers and partially thioaptamers may also be delivered as anintranasal form via use of a suitable intranasal vehicle. For dermal andtransdermal delivery, the aptamers and partially thioaptamers may bedelivered using lotions, creams, oils, elixirs, serums, transdermal skinpatches and the like, as are well known to those of ordinary skill inthat art. Parenteral and intravenous forms may also includepharmaceutically acceptable salts and/or minerals and other materials tomake them compatible with the type of injection or delivery systemchosen, e.g., a buffered, isotonic solution. Examples of usefulpharmaceutical dosage forms for administration of aptamers and partiallythioaptamers may include the following forms.

Capsules. Capsules may be prepared by filling standard two-piece hardgelatin capsules each with 10 to 500 milligrams of powdered activeingredient, 5 to 150 milligrams of lactose, 5 to 50 milligrams ofcellulose and 6 milligrams magnesium stearate.

Soft Gelatin Capsules. A mixture of active ingredient is dissolved in adigestible oil such as soybean oil, cottonseed oil or olive oil. Theactive ingredient is prepared and injected by using a positivedisplacement pump into gelatin to form soft gelatin capsules containing,e.g., 100-500 milligrams of the active ingredient. The capsules arewashed and dried.

Tablets. A large number of tablets are prepared by conventionalprocedures so that the dosage unit was 100-500 milligrams of activeingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams ofmagnesium stearate, 50-275 milligrams of microcrystalline cellulose, 11milligrams of starch and 98.8 milligrams of lactose. Appropriatecoatings may be applied to increase palatability or delay absorption.

Effervescent tablets. To provide an effervescent tablet appropriateamounts of, e.g., monosodium citrate and sodium bicarbonate, are blendedtogether and then roller compacted, in the absence of water, to formflakes that are then crushed to give granulates. The granulates are thencombined with the active ingredient, drug and/or salt thereof,conventional beading or filling agents and, optionally, sweeteners,flavors and lubricants.

Injectable solution. A parenteral composition suitable foradministration by injection is prepared by stirring 1.5% by weight ofactive ingredient in deionized water and mixed with, e.g., up to 10% byvolume propylene glycol and water. The solution is made isotonic withsodium chloride and sterilized using, e.g., ultrafiltration. Parenteraland intravenous forms may also include minerals and other materials tomake them compatible with the type of injection or delivery systemchosen.

Suspension. An aqueous suspension is prepared for oral administration sothat each 5 ml contain 100 mg of finely divided active ingredient, 200mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g ofsorbitol solution, U.S.P., and 0.025 ml of vanillin.

Mini-tabs. For mini-tablets, the active ingredient is compressed into ahardness in the range 6 to 12 Kp. The hardness of the final tablets isinfluenced by the linear roller compaction strength used in preparingthe granulates, which are influenced by the particle size of, e.g., themonosodium hydrogen carbonate and sodium hydrogen carbonate. For smallerparticle sizes, a linear roller compaction strength of about 15 to 20KN/cm may be used.

Kits. The present invention also includes pharmaceutical kits useful,for example, for the treatment of pathogenic infection or even a cancer.The kit will generally include one or more containers containing apharmaceutical composition with a therapeutically effective amount ofthe aptamers and/or partially thioaptamers disclosed herein. Such kitsmay further include, one or more of various conventional pharmaceuticalkit components, e.g., containers with one or more pharmaceuticallyacceptable diluents, as will be readily apparent to those skilled in theart. Printed instructions, either as inserts or as labels, indicatingquantities of the components to be administered, guidelines for mixtureand/or administration, may also be included in the kit.

The aptamers and partially thioaptamers and, optionally, one or morepotentiators may be mixed with a pharmaceutically acceptable carrier.The carrier may be a solid or liquid and the type is generally chosenbased on the type of administration being used. The active agent may becoadministered in the form of a tablet, capsule, liposome, as anagglomerated powder, in a liquid form or as a suppository.

Vaccines. The present invention includes vaccines for both active andpassive immunization. Immunogenic compositions, suitable for use as avaccine, include the modified thioaptamers of the present invention. Thethioaptamers are prepared in a manner disclosed herein. The vaccinesdisclosed herein are not the antigenic material, that is, they are notintended to cause an immune response, but rather, are include eitheralone or in combination with an antigen to “drive” or modify an immuneresponse by altering the activity of nuclear binding proteins,including, e.g.: NF-ATs, AP-1s, NF-IL6, NF-κB, HIV reversetranscriptase, Venezuelan Equine Encephalitis nucleocapsid (using an RNAthioaptamer), HepC IRES nucleic acid, protein(s) involved in CpG-induced“innate immunity,” and the like. As known to those in the immunologicalarts, the type of immunity, e.g., innate and/or adaptive, that isactivated (or deactivated) is a critical step in the immune response. Assuch, the thioaptamers may be under some circumstances acting as anadjuvant but in others will actually be a direct participant in theimmune response alone, that is, without addition of an antigen. Thethioaptamers may even be used to prime the immune system prior achallenge.

In operation, the thioaptamer will generally be extensively dialyzed toremove undesired small molecular weight molecules and/or lyophilized formore ready formulation into a desired vehicle. The preparation ofvaccines that include normal antigens are generally well understood inthe art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903;4,599,231; 4,599,230; 4,596,792; and 4,578,770, relevant portions ofthese incorporated herein by reference. Typically, such vaccines areprepared as injectables. Either as liquid solutions or suspensions:solid forms suitable for solution in, or suspension in, liquid prior toinjection may also be prepared. The preparation may also be emulsified.The active immunogenic ingredient is often mixed with excipients thatare pharmaceutically acceptable and compatible with the activeingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccines.

In vaccine form the thioaptamer may be administered, e.g., parenterally,by injection, for example, either subcutaneously, intraperitoneally,intranasally or into the lungs or even intramuscularly. Additionalformulations that are suitable for other modes of administration includesuppositories and, in some cases, oral formulations. For suppositories,traditional binders and carriers may include, for example, polyalkaleneglycols or triglycerides: such suppositories may be formed from mixturescontaining the active ingredient in the range of 0.5% to 10%, or even1-2%. Oral formulations include such normally employed excipients as,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,mixtures thereof and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10-95% of active ingredient,preferably 25-70%.

The thioaptamers may be administered directly to the aerodigestivesystem (the pulmonary system and/or digestive tract) of a patient by aninhaled aerosol. Delivery of drugs or other active ingredients directlyto a patient's lungs provides numerous advantages including: providingan extensive surface area for drug absorption; direct delivery oftherapeutic agents to the disease site in the case of regional drugtherapy; reducing the possibility of drug degradation in the patient'sintestinal tract (a risk associated with oral administration); andeliminating the need for repeated subcutaneous injections. Furthermore,delivery of the thioaptamers to the pulmonary system via aerosolinhalation may be used to deliver drugs systemically, as well as fortargeted local drug delivery for treatment of respiratory ailments suchas pathogenic infections (viral, bacterial and fungal) or even lungcancer or asthma. Aerosol devices for use with the present invention inthe clinical context include metered dose inhalers, dry powder inhalers,nebulizers and the like.

The thioaptamers may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts include those that are formedwith inorganic acid, e.g., sodium, potassium, ammonium, calcium, orferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, mixturesthereof and the like. The vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective. The quantity to be administered depends onthe subject to be treated, including, e.g., the capacity of theindividual's immune system to activate an innate immune response,synthesize antibodies or mount an effective cytotoxic T cell response,and the degree of protection desired. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner, however, suitable dosage ranges are of the order of a fewto several hundred micrograms active ingredient per vaccination.Suitable regimes for initial administration and booster shots are alsovariable, but are typified by an initial administration followed bysubsequent inoculations or other administrations. The manner ofapplication may be varied widely. Any of the conventional methods foradministration of a vaccine are applicable. These are believed toinclude oral application on a solid physiologically acceptable base orin a physiologically acceptable dispersion, parenterally, by injectionor the like. The dosage of the vaccine will depend on the route ofadministration and will vary according to the size of the host.

Various methods of achieving an additional or complementary adjuvanteffect for the thioaptamer may include, e.g., aluminum hydroxide orphosphate (alum), commonly used as 0.05 to 0.1 percent solution inphosphate buffered saline, admixture with synthetic polymers of sugars(Carbopol) used as 0.25 percent solution. When provided with a antigenicprotein, the thioaptamer may be aggregated with the antigen and othercomponents of the vaccine by heat treatment with temperatures rangingbetween 700 to 101° C. for 30 second to 2 minute periods. Examples ofaggregation include reactivating with pepsin treated (Fab) antibodies toalbumin, mixture with bacterial cells such as C. parvum or endotoxins orlipopolysaccharide components of gram-negative bacteria, emulsion inphysiologically acceptable oil vehicles such as mannide mono-oleate(Aracel A) or emulsion with 20 percent solution of a perfluorocarbon(Fluosol-DA) used as a block substitute may also be employed.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and one or more, usually at least aboutthree vaccinations. The vaccinations will normally be at from two totwelve week intervals, more usually from three to five week intervals.Periodic boosters at intervals of 1-5 years, usually three years, willbe desirable to maintain protective levels of the antibodies. The courseof the immunization may be followed by assays for antibodies for thesupernatant antigens. The assays may be performed by labeling withconventional labels, such as radionuclides, enzymes, fluorescers, andthe like. These techniques are well known and may be found in a widevariety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and3,949,064, as illustrative of these types of assays.

The thioaptamers may be used as part of a vaccine to regulate thedevelopment of Th1 or Th2 subsets in a subject or patient. In additionto in vivo modulation, the thioaptamers nay be used ex vivo to modifycells in vitro that are then administered to the subject. Moreparticularly, the thioaptamers disclosed herein may be used to modulatethe activity of a transcription factor (e.g., AP-1, NF-κB or NF-ATfamily members) that regulate innate or adaptive immune responses. Inone example the thioaptamer modulates the development of Th1 or Th2cells in the subject is modulated.

The thioaptamer vaccine may include more that one thioaptamer in orderto modulate the activity of additional transcription factors thatcontribute to regulating the expression of Th1- or Th2-associatedcytokines. In one embodiment, a stimulatory method includes a firstthioaptamer that modulated the activity of an AP-1 protein and a secondagent that modulates the activity of an NF-AT protein. The second agentmay be a thioaptamer or even an antigen.

The thioaptamer and the methods disclosed herein may be used tomanipulate Th1:Th2 ratios in a variety of clinical situations. Forexample, a thioaptamer may be provided that inhibits Th2 activation,which may be useful in allergic diseases, malignancies and infectiousdiseases. Conversely, the thioaptamer may be used to enhance Th2activation for treatment of autoimmune diseases and/or to improve organtransplantation.

The present inventors recognized that it is not possible to simplyreplace thiophosphates in a sequence that was selected for binding witha normal phosphate ester backbone oligonucleotide. Simple substitutionwas not practicable because the thiophosphates can significantlydecrease (or increase) the specificity and/or affinity of the selectedligand for the target. It was also recognized that thiosubstitutionleads to a dramatic change in the structure of the aptamer and hencealters its overall binding affinity. The sequences that werethioselected according to the present methodology, using as examples ofDNA binding proteins AP-1, NF-IL6 and NF-κB, were different from thoseobtained by normal phosphate ester combinatorial selection.

The present invention takes advantage of the “stickiness” of thio- anddithio-phosphate ODN agents to enhance the affinity and specificity to atarget molecule. In a significant improvement over existing technology,the method of selection concurrently controls and optimizes the totalnumber of thiolated phosphates to decrease non-specific binding tonon-target proteins and to enhance only the specific favorableinteractions with the target. The present invention permits control overphosphates that are to be thio-substituted in a specific DNA sequence,thereby permitting the selective development of aptamers that have thecombined attributes of affinity, specificity and nuclease resistance.

In one embodiment of the present invention, a method of post-selectionaptamer modification is provided in which the therapeutic potential ofthe aptamer is improved by selective substitution of modifiednucleotides into the aptamer oligonucleotide sequence. An isolated andpurified target binding aptamer is identified and the nucleotide basesequence determined. Modified achiral nucleotides are substituted forone or more selected nucleotides in the sequence. In one embodiment, thesubstitution is obtained by chemical synthesis using dithiophosphatenucleotides. The resulting aptamers have the same nucleotide basesequence as the original aptamer but, by virtue of the inclusion ofmodified nucleotides into selected locations in the sequences, improvednuclease resistance and affinity is obtained.

RNA and DNA oligonucleotides (ODNs) can act as “aptamers,” (i.e., asdirect in vivo inhibitors selected from combinatorial libraries) for anumber of proteins, including viral proteins such as HIV RT (Burke etal., 1996; Chen & Gold, 1994; Green et al., 1995; Schneider et al.,1995) and transcription factors such as human NF-κB (Bielinska et al.,1990; Lebruska & Maher, 1999; Lin et al., 1998; Morishita et al., 1997;Sharma et al., 1996). Decoy ODNs were developed to inhibit expressionfrom CRE and AP-1 directed transcription in vivo and inhibit growth ofcancer cells in vitro and in vivo (Park et al., 1999). These studies andothers (Boccaccio et al., 1998; Cho-Chung, 1998; Eleouet et al., 1998;Jin & Howe, 1997; Mann, 1998; Morishita et al., 1995; Morishita et al.,1998; Osborne et al., 1997; Tomita et al., 1997) have demonstrated thepotential of using specific decoy and aptamer ODNs to bind to variousproteins, serve as therapeutic or diagnostic reagents, and to dissectthe specific role of particular transcription factors in regulating theexpression of various genes. In contrast to antisense agents, duplexaptamers appear to exhibit few if any non-specific effects.

Among a large variety of modifications, S-ODN and S₂-ODN render theagents more nuclease resistant. The first antisense therapeutic druguses a modified S-ODN (CIBA Vision, A Novartis Company). The S₂-ODNsalso show significant promise, however, the effect of substitution ofmore nuclease-resistant thiophosphates cannot be predicted, since thesulfur substitution can lead to significantly decreased (or increased)binding to a specific protein (Milligan, J. F. and Uhlenbeck, O. C.(1989) and King et al., 2002 as well as structural perturbations (Volket al., 2002) and thus it is not possible to predict the effect ofbackbone substitution on a combinatorially selected aptamer. Hence, thepresent inventors recognized that selection should be carried outsimultaneously for both phosphate ester backbone substitution and basesequence.

Phosphorodithioate analogs have been synthesized to produce an importantclass of sulfur-containing oligonucleotides, the dithiophosphateS₂-ODNs. These dithioates include an internucleotide phosphodiestergroup with sulfur substituted for both nonlinking phosphoryl oxygens, sothey are both isosteric and isopolar with the normal phosphodiesterlink, and are also highly nuclease resistant. One group showed highlyeffective protection of the dithioate against degradation by endogenousnucleases after 58% backbone modification. Significantly, the S₂-ODNs,in contrast to the phosphoramidite-synthesized monothiophosphate(S-ODNs), are achiral about the dithiophosphate center, so problemsassociated with diastereomeric mixtures (Lebedev & Wickstrom, 1996) arecompletely avoided. The S₂-ODNs and the S-ODNs, are taken up efficientlyby cells, especially if encapsulated in liposomes.

Thiophosphate aptamers or thioaptamers are capable of specifically andnon-specifically binding to proteins. Importantly, it has been observedby the present inventors that sulfurization of the phosphoryl oxygens ofoligonucleotides often leads to their enhanced binding to numerousproteins (Gorenstein, 1994). The dithioate agents, for instance, appearto inhibit viral polymerases at much lower concentrations than do themonothiophosphates, which in turn are better than the normal phosphates,with K_(d)'s for single strand aptamers in the nM to sub-nM range forHIV-1 RT (Marshall & Caruthers, 1993) and NF-κB (Yang et al., 2002, Kinget al. 2002). For HIV-1 RT, dithioates bind 28-600 times more tightlythan the normal aptamer oligonucleotide or the S-analogue. Sequence isalso important, as demonstrated by the observation that a 14-ntdithioate based on the 3′ terminal end of human tRNA^(Lys)(CTGTTCGGGCGCCA) (SEQ ID NO.: 10) complementary to the HIV primerbinding site is a more effective inhibitor (ID₅₀=4.3 nM) than simplydithioate dC₁₄ (SEQ ID NO: 59) (ID₅=62 nM) by an order of magnitude(Marshall & Caruthers, 1993).

Oligonucleotides with high monothio- or dithiophosphate backbonesubstitutions appear to be “stickier” towards proteins than normalphosphate esters, an effect often attributed to “non-specificinteractions.” One explanation for the higher affinity of thethiosubstituted DNAs is the poor cation coordination of the polyanionicbackbone (Cho et al., 1993, Volk et al., 2002) sulfur, being a softanion, does not coordinate as well to hard cations like Na⁺, unlike thehard phosphate oxyanion. The thiosubstituted phosphate esters then actas “bare” anions, and since energy is not required to strip the cationsfrom the backbone, these agents appear to bind even more tightly toproteins.

Even in specific protein-nucleic acid contacts, sulfurization of theinternucleotide linkages can lead to enhanced binding (Marshall &Caruthers, 1993; Milligan & Uhlenbeck, 1989) (or to decreased affinity).The enhanced binding is very important, since most of the directcontacts between DNA-binding proteins and their binding sites are to thephosphate groups (Otwinowski et al., 1988) (Chen et al., 1998; Ghosh etal., 1995; Muller et al., 1995). The present invention takes advantageof this chemical “stickiness” to enhance the specificity and affinity ofthio- and dithiophosphate agents for a protein target. It was necessary,however, to optimize the total number of thioated phosphates to decreasenon-specific binding to non-target proteins and thus enhance only thespecific favorable interactions with the target protein. Also,thiosubstitution can also perturb the structure of the duplex (Cho etal., 1993) (Volk et al, 2002) although monothiophosphates substituted inthe DNA strand of DNA/RNA hybrids do not appear to have dramaticallyaltered duplex structures (Bachelin et al., 1998; Gonzalez et al.,1995). The present invention uses sequence-based, structure-based andcombinatorial methods to identify both sequences and thiophosphatesubstitution patterns to develop thioaptamers that retained the highestspecificity and affinity in binding to target proteins. The use ofpartial thiophosphate substitution resulted in aptamer that were morestable in vivo.

In vitro combinatorial selection of thiophosphate aptamers may be usedwith the present invention. A recent advance in combinatorial chemistryhas been the ability to construct and screen large random sequencenucleic acid libraries for affinity to proteins or other targets (Eklandet al., 1995; Gold et al., 1997; Tian et al., 1995). The aptamer nucleicacid libraries are usually selected by incubating the target (protein,nucleic acid or small molecule) with the library and then separating thenon-binding species from the bound. The bound fractions may then beamplified using the polymerase chain reaction (PCR) and subsequentlyreincubated with the target in a second round of screening. Theseiterations are repeated until the library is enhanced for sequences withhigh affinity for the target. However, agents selected fromcombinatorial RNA and DNA libraries have previously always had normalphosphate ester backbones, and so would generally be unsuitable as drugsor diagnostics agents that are exposed to serum or cell supernatantsbecause of their nuclease susceptibility. The effect of substitution ofnuclease-resistant thiophosphates cannot be predicted, since the sulfursubstitution can lead to significantly decreased (or increased) bindingto a specific protein (Milligan & Uhlenbeck, 1989).

The present invention have described the combinatorial selection ofphosphorothioate oligonucleotide aptamers from random orhigh-sequence-diversity libraries, based on tight binding to the target(e.g. a protein or nucleic acid) of interest, relevant portions of whichare incorporated herein by reference. An in vitro selection approach forRNA thioaptamers has also been described Ellington and co-workers(Jhaveri et al., 1998).

One approach used by the inventors is a hybrid monothiophosphatebackbone. Competition assay for binding CK-1 42-mer aptamers wereconducted. In standard competitive binding assays, ³²P-IgκB promoterelement ODN duplex was incubated with recombinant p50 or p65 andcompetitor oligonucleotide. The reactions were then run on anondenaturing polyacrylamide gel, and the amount of radioactivity boundto protein and shifted in the gel was quantitated by direct counting.

A combinatorial library was created by PCR, using an appropriatedNTP(αS) in the Taq polymerization step. A combinatorial thiophosphateduplex and single stranded (ss) libraries was screened successfully forbinding to a number of different protein and nucleic acid targets,including NF-IL6, NF-κB, HIV reverse transcriptase, Venezuelan EquineEncephalitis nucleocapsid (using an RNA thioaptamer), HepC IRES nucleicacid, and others, including a protein involved in CpG-induced “innateimmunity.” Briefly, a filter binding method was used that was modifiedto minimize non-specific binding of the S-ODNs to the nitrocellulosefilters. A column method may also be used in which the target iscovalently attached to a column support for separation as well. Theduplex, ssDNA and/or ssRNA S-ODN's are eluted from the filter under highsalt and protein denaturing conditions. Subsequent ethanol precipitationand for the duplex DNA S-ODNs, another Taq polymerase PCR thiophosphateamplification provided product pools for additional rounds of selection(for RNA thioaptamers RT and T7 polymerase were used). To increase thebinding stringency of the remaining pool of S-ODNs in the library andselect higher-affinity members, the KCl concentration was increased andthe amount of protein in subsequent rounds was reduced as the iterationnumber increased. After cloning, the remaining members of the librarywere sequenced, which allowed for “thioselect”™ simultaneously for bothhigher affinity and more nuclease-resistant, “thioaptamer”™ agents. Thethioselect method has been used to isolate a tight-binding thioaptamerfor 7 of 7 targets tested.

NF-κB thioaptamers were created using thioselect for both in vitrothioselection as well as rational design of thioaptamers against NF-κB(Gorenstein at al., 1999a,b; 2001, 2002; King at al., 2002). Sharma, etal. demonstrated previously effective aptamer inhibition of NF-κBactivity. They further achieved inhibition of NF-κB in cell cultureusing S-ODN duplex decoys with NF-κB binding consensus-like sequence(GGGGACTTCC) (SEQ ID NO: 601. The present inventors used the “CK-1”42-mer duplex oligonucleotide identified by Sharma et al. (note: boththe present inventors and Sharma et al.'s S-ODN duplex was chemicallysynthesized by sulfur oxidation with phosphoramidite chemistry and thuscontains in principle 2⁸² or 10²⁴ different stereoisomers!). Thewild-type CK-1 duplex sequence contains 3 tandem repeats of a 14-merNF-κB consensus-like sequence (5′-CCA GGA GAT TCC ACC CAG GAG ATT CCACCC AGG AGA TTC CAC 3′) (SEQ ID NO.: 11).

S-ODN CK-1 monothioate aptamers were made because it was unlikely thatthe phosphodiester form is appropriate for therapeutics or diagnosticsbecause of its short half-life in cells, cell extracts and serum. Thephosphorothioate and dithioate internucleoside modifications aretherefore needed. Using recombinant protein homodimers of p50, p65, andc-Rel, the present inventors confirmed that the CK-1 sequence could bindto and compete for binding to p65 homodimer, but not p50/p50, instandard electrophoretic mobility shift assays (EMSA)(data not shown).In contrast to the fully substituted phosphorothioate, the CK-1 aptamerinhibited p65/p65 and p50/p50 equally; confirming that S-ODNs with largenumbers of phosphorothioate linkages are “sticky” and tend to bindproteins non-specifically. The present inventors also found that if thenumber of phosphorothioate linkages is decreased to only 2-4,specificity can be restored, but binding is not enhanced. Therefore, theoriginal publications described only the specificity of thephosphodiester oligonucleotides and did not address the problem ofaltered specificity of the phosphorothioates.

Changing from purified recombinant proteins to cell culture andextracts, the situation is further complicated by the presence of theother cellular components, besides the presence of other naturallyoccurring NF-κB homo- and heterodimers. When the present inventorsattempted to repeat the binding inhibition studies of others using cellextracts, unexpected difficulties were encountered. It was found thatthe diester form of the CK-1 aptamer does not compete effectively forNF-κB binding in cell extracts derived from two different cell lines:the 70Z pre-B cell line and the RAW 264.7 mouse macrophage-like line.The heterodimers in these cells either do not bind the CK-1 sequencetightly enough, or it is bound by other cellular components. Publishedreports describing CK-1 did not present data using cell extracts,perhaps due to similar difficulties (Sharma et al., 1996). Therefore,even sequences with good binding and specificity in the diester form,when fully thiophosphate-substituted, lose their sequence specificity.Thus, this stickiness makes the characterization of fully thioatedaptamers in vitro not necessarily predictive of their activities invivo.

TABLE 1 DNA Sequences from p50 Selection Group 1 Sequences (n = 16)Number of Clones CTG TGT TCT TGT GCC GTG TCC C 6/22 (SEQ ID NO.: 12) CTGTGT TCT TGT GTC GTG TCC C 4/22 (SEQ ID NO.: 13) CTG TGT TCT TGT GTC GTGCCC C 3/22 (SEQ ID NO.: 14) CCG TGT TCT TGT GCC GTG TCC C 2/22 (SEQ IDNO.: 15) CCG TGT TCT TGT GTC GTG TCC C 1/22 (SEQ ID NO.: 16)

TABLE 2 DNA Sequences from p65 Selection Number of Clones Group 1Sequences (n = 8) CGG GGT GTT GTC CTG TGC TCT CC 7/16 (SEQ ID NO.: 17)CGG GGT GTT CTC CTG TGC TCT CC 1/16 (SEQ ID NO.: 18) Group 2 Sequences(n = 4) CGG GGT GGT GTG GCG AGG CGG CC 2/16 (SEQ ID NO.: 19) CGG GGT GGTGCG GCG AGG CGG CC 1/16 (SEQ ID NO.: 20) CGG GGT GTG CTG CTG CGG GCG GC1/16 (SEQ ID NO.: 21) CGG GGT GTG CTG CTG CGG GCG GC 1/16 (SEQ ID NO.:22)

Thioselection against NF-κB (p50:p50, p65:p65). As described in King, etal. (2002) a unique thiophosphate duplex library was screened forbinding to the p50 homodimer. Thioselection was repeated through 15rounds to enrich for sequences that bind to p50 with high affinity. DNAsequences of multiple clones were analyzed from the initial, 2nd,6^(th), 10^(th) and 15^(th) round libraries. A striking convergence ofthe DNA sequences was observed by round 15. Of the 22 clones analyzed,16 had a highly similar sequence (Table 1). A thioaptamer representingthis sequence was generated by PCR amplification using a biotinylatedreverse primer. Binding studies were conducted using a chemiluminescentEMSA, which uses a biotinylated thioaptamer. The biotinylatedthioaptamer binds tightly to p50; the sequences are different from thoseobtained for in vitro combinatorial selection against p65 homodimers(Table 2). The chemically synthesized phosphorothioate aptamers are adiastereomeric mixture of both Rp and Sp configurations. Thethioaptamers bind and compete for the same NF-κB site as the knownpromoter element IgκB (K_(d)=78.9±1.9 nM for a Rel A-selectedthioaptamer, and 19.6±1.25 nM for a p50-selected thioaptamer). Thenormal phosphate ester backbone version of the Rel A selected aptamerbinds Rel A with a K_(d) of 249.1±1.8 nM. The p50 dimer-selected chiralthioaptamer binds to p50 with affinities below 5 nM under conditionswhere no binding to p65 is observed. Similarly, the p65 dimer-selectedchiral thioaptamer binds to p65 dimers with affinities below 5 nM underconditions where no binding to p50 is observed.

These EMSA binding studies demonstrated that the enhanced affinity canbe attributed to the presence of sulfur. Collectively, these resultsfurther demonstrate the feasibility of the thioaptamer selectiontechnology as a method for producing specific, high-affinity ligands toproteins. It was also demonstrated that the chemically synthesized(mixed diastereomer) thioaptamers bind tightly in cell nuclear extractsto both the p50:p65 heterodimer and p50:p50 homodimer. However, theenzymatically synthesized, chiral thioaptamer selected against the p50homodimer only binds to p50:p50 in nuclear extracts (Fennawald, et al,unpublished; King, et al., 2002; Gorenstein, patents pending, 1999a, b,2001). Remarkably, for the p50 homodimer the selection sequence appearsto contain a pseudo-palindrome, suggesting that 2 dimers may be bindingto the 22-mer sequence:

CTGTG PyT (CT) T G* T (G) TPy (SEQ ID NO.: 23) GTGTC CC

Dithiophosphate Aptamers Binding to Proteins. S₂-ODN CK-14 dithioateaptamers were also isolated. The CK-14 14-mer duplex was alsosynthesized with some strategically placed dithioate linkages (both ofthe non-bridging oxygens are replaced by sulfurs). As noted by thepresent inventors, strategic dithioate linkage ODNs have exhibitsignificant differences, as they have altered binding specificity, andlack the extreme “stickiness” of the fully thioated aptamer. With anincreasing number of dithioate substitutions in the same sequence,binding by the S₂-ODN increases dramatically (data not shown). One ofthe tightest-binding dithioaptamer (XBY-6) contains 6 dithioate linkageson the two strands. Significantly, the XBY-6 aptamer also binds to asingle NF-κB dimer in cell extracts (data not shown), while the standardphosphodiester ODN shows no NF-κB-specific binding in extracts. Thus,the present inventors succeeded in synthesizing a thioate backbonemodification which for the first time increases the specific binding ofthe oligonucleotide to NF-κB above that to other cellular proteins (Yanget al., 1999). In standard competitive binding assays, the ³²P-IgκBpromoter element ODN was incubated with recombinant p65 and varyingamounts of XBY decoy competitor. The relative binding ability of theunlabeled ODNs was determined by the concentration needed to competeeffectively with the standard labeled ODN. XBY1 through 6 correspond toCK-14 aptamers with 1 though 6 dithiophosphate substitutions,respectively (Yang, et al., 1999).

ODN aptamer was incubated with 70Z/3 cell nuclear extract in thepresence or absence of anti-p50 antibody. Protein-bound ODN duplex wasseparated on a standard gel. XBY-6 shifts one complex in nuclearextracts from a 70Z/3 pre-B cell line. By using specific antibodies tosupershift the complex, p50 was identified as one component of thecomplex, which may be a complex that include a p50 or p105 dimer, or ap50 (or p105)-containing heterodimer. Since XBY-6 binds more tightly top50/p50 than p65/p65, the shifted band is likely to represent the p50homodimer. The band did not co-migrate with either the p50/p50 orp50/p65 bands, but the change in the altered chemical structure changesthe mobility of the ODN. Only one major band is seen, however, eventhough the lysate contains at least two major distinguishable NF-κBcomplexes (p50 homodimers and p50/p65 heterodimers).

These results demonstrate the use of aptamers having altered bindingspecificity and affinity by substituting only a limited number ofinternucleoside linkages, that is, a portion of the internucleosidelinkages. The partially-modified aptamer was used to distinguish amongvarious NF-κB dimers within the cell. The IgκB standard ODN does notshow such specificity. Therefore, this modified thioaptamer may be usedto bind to a single NF-κB dimer within cell supernatants and eveninactivate target dimers within whole cells and animals. It was alsofound that when guinea pigs were injected with LPS to induceinflammatory response and XBY-6, an increase in the levels of TNF-α wasobserved above that when the animals were injected with LPS alone. Inanimal macrophage extract studies, it was found that XBY-6 eliminated asingle p50 (or p105) dimer band on EMSAs. Since the p50 homodimerappears to be a transcriptional inhibitor of the immune response, thesedata demonstrate the ability to target a single protein within liveanimals, and the feasibility of altering the binding specificity bysubstituting only a limited number of internucleoside linkages(Gorenstein, et al. patents pending, 1999a, b; 2001, 2002). Using themodified thioaptamer a 1:1 binding stoichiometry of p65 to the 22 merbinding site known as IgκB with a K_(d) near 4 nM. For onedithiophosphate aptamer, XBY-6, a binding affinity to p65 homodimer of1.4 nM vs. sub-nM to p50 was demonstrated.

Various thioaptamers have been made and isolated using the presentinvention that can distinguish among various NF-κB dimers within thecell. One of these decoys was able to bind to a single NF-κB dimer incell extracts or within a cell in either cell culture or animal studies.These results point to the importance of using modified thiophosphatecombinatorial selection methods to identify minimally substitutedthioated oligonucleotides with high affinity, high binding specificityand increased nuclease resistance in vitro and in vivo.

Phosphorodithioate and phosphorothioate aptamers via split synthesiscombinatorial selection. The identification of specific S-ODN and S₂-ODNthioaptamers that bind proteins based upon in vitro combinatorialselection methods is limited to substrates only accepted by polymerasesrequired for reamplification of selected libraries by the polymerasechain reaction (PCR). Another disadvantage of using the polymerizationof substituted nucleoside 5′-triphosphates into ODN aptamers are therestrictions on the choice of P-chirality by the enzymaticstereospecificity. For example, it is known that[S_(P)]-diastereoisomers of dNTP(αS) in Taq-catalyzed polymerizationsolely yield [R_(P)]-phosphorothioate stereoisomers (Eckstein, 1985).Therefore, using current methods it is not possible to select[S_(P)]-phosphorothioate stereoisomers along with achiral S₂-ODNanalogous since both [Rp]-diastereoisomers of dNTP(αS) and nucleosidedNTP(αS₂) are not substrates of polymerases. Additionally, these invitro combinatorial selection methods require many iterative cycles ofselection and reamplification of the bound remaining members of thelibrary by the PCR, which are quite time consuming, although automationof this in vitro selection is possible.

What is needed are methods that permit the isolation of, e.g.,individual aptamer:protein complexes without the need for repeatediterative cycles of selection and reamplification of likely bindingtargets. Also needed are systems that permit the creation, isolation,sequencing and characterization of making [S_(P)]-phosphorothioatestereoisomers along with achiral S₂-ODN analogs. To overcome theselimitations of the in vitro combinatorial selection methods, the presentinventors developed a one-bead, one-compound library made by using asplit synthesis method to create an alternative to in vitrocombinatorial selection methods. One-bead library systems have been usedfor organic molecules (Felder, (1999)), peptides (Lam, et al., 1991,1995; Lam, 1995), and oligosaccharide libraries (Zhu and Boom, 1998;Liang, et al., 1996; Hilaire and Meldal, 2000). A one-beadone-oligonucleotide (one-ODN) (e.g., O-ODN, S-ODN, S₂-ODN, both DNA orRNA) may be used in conjunction with combinatorial library selectionmethodology used to identifying a specific oligonucleotide aptamer thatbinds to specific proteins or other molecules (Yang, et al., 2002;Gorenstein, et al., U.S. patent applied).

Furthermore, the method may use S₂-ODN reagents with sulfurs replacingboth of the non-bridging phosphate oxygens that are isosteric andisopolar with the normal phosphorodiester and are particularlyadvantageous for binding and screening. Importantly, S₂-ODNs are achiralabout the dithiophosphate center, which eliminated problems associatedwith diastereomeric mixtures generally obtained for the chemicallysynthesized S-ODN. The split synthesis approach disclosed herein hasbeen used for the construction of O-ODN, S-ODN, S₂-ODN and RNAbead-based aptamer and thioaptamer libraries (Gorenstein et al, USPatents pending, 1999a, b, 2001, 2002; awarded, 2002; Yang et al.,2002). In this procedure each unique member of the combinatorial libraryis attached to a separate support bead. Targets that bind tightly toonly a few of the 10⁴-10₈ different support beads can be selected bybinding the target protein to the beads and then identifying which beadshave bound target by immunostaining techniques or direct staining of thetarget or SELDI MS (see below). The present methodology permits rapidscreening and identification of modified thioaptamers that bind toproteins such as NF-κB using a novel PCR-based identification tag of theselected bead.

To introduce many copies of a single, chemically pure S-ODN thioaptameronto each bead, a “mix and separate” split synthesis method was used. Atwo-column DNA synthesizer was used simultaneously for construction ofthe library. The normal phosphate backbone linkages were carried outusing standard phosphoramidite monomers via oxidation in column 1, whilethe phosphorothioate linkages were carried out using standardphosphoramidite monomers via sulfurization in column 2. Dithioate areintroduced by using thiophosphoramites with sulfur oxidation. Twosequences of the same length are programmed for each column and aredesigned such that the bases are different at every equal position notonly for diversifying base compositions but also for coding a phosphate,phosphoromonothioate/dithioate.

For example, on an Expedite 8909 DNA synthesizer with dual columns, ontocolumn 1 a phosphoramidite (for example: C) is coupled to the bead andafter completion of oxidation, the resulting product is nucleotide (C)with a phosphotriester linkage. On column 2 a nucleosidephosphorothioate is introduced with a different base (T for example).The two columns are mixed and resplit and in the second cycle,additional phosphoramidites or phosphorothioamidites are introduced,followed by oxidation and sulfurization reactions individually in column1 and column 2. After additional coupling steps and after split/poolsynthesis is carried out, the end products comprise a combinatoriallibrary of thioaptamers with varying monothioate, dithioate or normalphosphate ester linkages at varying positions along the ODN strand. Oncompletion of the automated synthesis, the column is removed from thesynthesizer and dried with argon. The bead bound fully protected ODNsare treated with 1 ml of concentrated ammonia for 1 h at roomtemperature, incubated in a 55° C. oven for 15-16 h, removed from theoven and cooled to room temperature. Importantly, after deprotection,with this coupling scheme with a non-cleavable hexaethyleneglycollinkers. Linker attaching the first phosphoramidite (15 or 70 μm beadsprovided by ChemGenes), the thioaptamers are still covalently attachedto the beads after complete deprotection. Thus, each bead contains asingle sequence with a specified backbone modification that isidentified by the base.

For example, this scheme was used to synthesize libraries of 4096 (2¹²)different thioaptamers attached to beads, each bead containing a uniquethioaptamer. This library consisted of a 22-nucleotide “random” sequence(12 split/pool steps) flanked by 15 nucleotide defined primer regions atthe 5′ and 3′ ends (Yang, et al., 2002). A phosphorothioate linkage wasintroduced on every other base in column 2, following the “split andpool” approach. The single-stranded 52-mer S-ODN random library wasconverted to double-stranded DNA by Klenow DNA polymerase 1 (Promega)reaction in the presence of DNA polymerase buffer, dNTP mix anddownstream primer. Therefore, the one strand of the duplex potentiallycontained S-ODN modifications and the other complementary strand werecomposed of ODN. A duplex DNA library in which both strands containS-ODN modifications could also be generated using a Klenow reaction withno more than three dNTP (α)S.

The dsDNA thioaptamer library beads were screened for the ability tobind the NF-κB p50/p50 dimer labeled with the Alexa Fluor 488 dye(Molecular Probes). After initial binding of protein, the beads werethoroughly washed with PBS with 0.1% Tween 20 to minimize nonspecificbinding. Typically, a few positive beads were intensely stained whenviewed by fluorescence, while the majority of the beads remainedunstained as (data not shown). With the aid of a micropipette coupled toa micromanipulator, the intensely stained beads were retrieved. Onlyhighly positive beads from several thousand were found using thismethod. As described below, multicolor flow cytometry and cell/beadsorting was used to automate the selection process to select thetightest binding thioaptamer-protein complexes.

Sequencing may also be obtained directly from the bead. Eachindividually selected bead was washed thoroughly with 8 M urea (pH 7.2)to remove the protein and was directly used for the “one-bead one-PCR”amplification using the 5′ and 3′ end primers. The PCR product wascloned using the TA Cloning procedure (Invitrogen) and sequenced on anABI Prism 310 Genetic Analyzer (Applied Biosystems). The fourthioaptamers listed in Table 3 were obtained from the library. Forverification of these results, the S-ODN, 5′-CtGTGAGtCGACTgAtGaCGGt-3′(SEQ ID NO.: 61) (small letters represent location of3′-monothiophosphates), was synthesized independently on thenon-cleavable linker bead support, hybridized with its complementary ODNand then mixed again with the p50/p50 protein labeled with the AlexaFluor 488 dye. The fluorescence intensity of all of the beads viewedunder the fluorescence microscope was qualitatively similar to theintensity of the selected bead containing this sequence within thecombinatorial library. These results demonstrate that the primer regionsdo not contribute to the binding of p50/50. Furthermore, it was foundthat not only normal monothio-ODN on the beads but also dithio-modifiedbead-bound sequences could be sequenced directly from thedithiophosphate combinatorial library. Thus, the split synthesis hasbeen used to create a “one-bead-one sequence” ODN and that PCR can beused to identify an S-ODN bound to a bead (Yang et al., 2002; Gorensteinet al, US & Foreign Patents pending, 1999 a, b, 2001, 2002).

Bead-based thioaptamer library screen. Aliquots of S-ODN beads bound toNF-κB p50/p50 homodimer protein labeled with the Alexa Fluor 488 dyeviewed under light microscopy. The same beads viewed under fluorescencemicroscopy, in which a positive green bead stained with Alexa Fluor 488dye were easily identified in a background of many hundreds ofnonreactive beads. Single positive bead can easily be retrieved with ahandheld micropipette under fluorescence microscopy.

Although the beads were screened against a target protein labeled with afluorescent dye, the beads have also been screened directly against cellextracts as well. The binding of the NF-κB to a specific sequence can bedetected using a primary anti-NF-κB antibody such as anti-P50 (RabbitIgG antibody, Santa Cruz Biotechnology, Inc.) followed by a secondaryantibody conjugated with Alexa Fluor 488 (goat anti-rabbit IgG fromMolecular Probes). Beads that included the XBY-6 oligonucleotide werescreened against WI-38 VA13, an SV40 virus-transformed humanfibroblastic cell line extract by similar fluorescent microscopy.

Other bead-based thioaptamer libraries. Combinatorial thioaptamer beadlibraries of over 10⁶ different sequences have also been readilyprepared. The present inventors have synthesized successfully a monothioRNA library (2¹⁵=32768) (Gorenstein, et al., patent pending, 2002).Thus, standard phosphoramidite (DNA and RNA) chemistry was used for thethioaptamer. RNA library. A 0.5 M 1H-tetrazole in acetonitrile was usedas DNA activator. A 0.5 M solution of DCI (dicyanoimidazole) inacetonitrile was used as RNA activator. The libraries were prepared on a1 μmole scale of polystyrene beads (66-70 μm). The downstream andupstream primers, 5′-d(GGATCCGGTGGTCTG)-3′ (SEQ ID NO: 26) and5′-d(CCTACTCGCGAATTC)-3′ (SEQ ID NO: 27) were synthesized in parallel ona two-column DNA synthesizer (Expedite 8909, Applied Biosystems).Following the 5′-primer, the sequences programmed on the synthesizer forthe combinatorial mono RNA library were5′-r(GA*UC*CU*GA*AA*CU*GU*UU*UA*AG*GU*UG*GC*CG*AU*C)-3′ (SEQ ID NO.: 24)on column 1 and 5′-r(cU*aG*gA*cU*uG*gC*aC*aA*cC*gU*cA*cA*cU*gC*uA*u)-3′(SEQ ID NO.: 25) on column 2. The 3′-primer sequence completed the61-mer programmed on the synthesizer. A “split and pool” occurred ateach position indicated by an asterisk in order to synthesize thecombinatorial region for the monothio RNA. The lower case letterindicates a 3′-thioate linkage, the upper case letter indicates a3′-phosphate linkage. The coupling yield was typically upwards of 98.5%as determined by the dimethoxytrityl cation assay (DNA couplings aretypically >99%/nt). Sulfurization chemistry utilized the Beaucagereagent. The fully protected monothio RNA combinatorial library with thenon-cleavable linker beads were treated with 4 ml of a mixture of 3:1(v/v) (28%) NH₃: EtOH at 39° C. for 21 hrs. The beads were centrifuged,the supernatant was removed and the solid support was washed withdouble-distilled water. After lyophilization the solid support wastreated with 2 ml of triethylamine trihydrofluoride (TEA-3HF) for 20 hrsat room temperature. Again, the beads were centrifuged, the supernatantwas removed and the solid support was washed with double-distilledwater. RT PCR and TA cloning confirmed the successful synthesis of thessRNA thioaptamer library.

TABLE 3 Sequences of thioaptamers selected from split synthesis (smallletters indicate thiophosphate 3′ to base). 5′-tGTGcAGGGACTgAtGaCGGt-3′(SEQ ID NO.: 6) 5′-CtGTGCatCGAaGTTtGCAtTt-3′ (SEQ ID NO.: 7)5′-AtGcAcAtCtCaGgAtGaCGGt-3′ (SEQ ID NO.: 8)5′-AGTTGcAGGtCaGgACCCAtTt-3′ (SEQ ID NO.: 9)

Flow cytometry sorting of thioaptamer bead-based library. The presentinventors have also demonstrated the successful application of highthroughput/multi-color flow cytometry and bead sorting to screen aptamerbead libraries for those beads which bind to, e.g., a target protein(Gorenstein, et al., patent pending, 2002). Modifications were made to acustom-built flow cytometer to make it more amenable to beadidentification and isolation. For example, bead fluorescence and forwardscatter were the two parameters chosen for real-time characterization ofeach aptamer bead passing the first sort point of the custom-built flowcytometer/sorter. Other scanning and sorting parameters may be used toselect, isolate, view, designate, characterize, etc. the beads through aflow cytometer.

In operation, “positive” beads (contain thioaptamer-bound targetprotein, the target protein was fluorescent-labelled with Alexa 488 dye)were easily sorted from negative beads. Flow cytometry may be used toreplace, e.g., visual fluorescence microscope identification of beadscontaining bound target protein and the need to isolate the individual“positive” beads with the micromanipulator described previously. Theflow-sorted “positive” beads can then be subjected to, e.g., one-beadPCR to identify the thioaptamer that binds the target protein.

TABLE 4 Population Statistics for bead sorting, WinList analyses (alldata were color-compensated) Sample Total Region % Gate FIG. 6A:CONTROL.FCS R1: Autofluorescent Beads 10000 9530 95.3 FIG. 6B. FCS R2:p50 Alexa 488 Positive Beads 10000 35 0.35 FIG. 6C. FCS R3: p65 PEPositive Beads 20000 3488 17.44 FIG. 6D. FCS R1: Autofl. Beads & CarrierBeads 1000000 963321 96.33 R2: p50 Alexa 488 Positive Beads 1000000 3540.04 R3: p65 PE Positive Beads 1000000 935 0.09

Fluorescence sorting was also used to demonstrate the use of theone-bead, one-ODN:protein system using dual color sorting. The IgκBdsDNA consensus sequences were immobilized onto 15-20 micron polystyrenemicrospheres. The DNA bound beads were then incubated with purified p50and p65 proteins, respectively. DNA transcription factor complexes weredetected with primary antibodies specific for the p50 and p65 proteinsfollowed by an additional incubation with Alexa 488-conjugated secondaryantibody for p50 and PE-conjugated secondary antibody for p65. The beadswere viewed by fluorescent microscopy and then analyzed on the MCU'sHiReCS system. A Control Fluorescent Cell Sort (CONTROL.FCS) shows theautofluorescent microspheres in the negative control sample where thebeads were unbound. The majority of the beads in the “debris” populationwere the 0.8 micron carrier beads that were used to bring up the volumeof the samples since the beads were at a very low dilution.

Innate Immunity Toll-Like Receptor Signaling. In another embodiment ofthis invention, the present inventors developed thioaptamers thatenhance the innate immune response by targeting the Toll-like receptor(TLR) family in mammals, which is a family of transmembrane proteinscharacterized by multiple copies of leucine rich repeats in theextracellular domain and IL-1 receptor motif in the cytoplasmic domain(Akira et al., 2001; Medzhitov, 2001). The TRL family is aphylogenetically conserved mediator of innate immunity that is essentialfor microbial recognition. Ten human homologs of TLRs (TLRI-10) havebeen described. By using a BLAST search, Hemmi et al., 2000, haveidentified and subsequently isolated a cDNA coding for TLR9. Geneknockout experiments suggest that TRL9 acts as a receptor forunmethylated CpG dinucleotides in the bacterial DNA. Human and mouseTLR9 share an overall amino-acid identity of 75.5%. TLR9 is highlyexpressed in spleen (Krieg, 2002).

The immunostimulatory properties of bacterial DNA appears to be relatedto short six base sequences called CpG motifs that have the generalstructure of two 5′ purines, an unmethylated CpG motif, and two 3′pyrimidines (Krieg, 2002). Though such sequences rarely appear inmammalian DNA due to CpG suppression and methylation of cytosinenucleotides, they are relatively abundant in bacterial DNA, occurring atthe expected frequency (1 in 16) and in unmethylated form. Indeed,studies have found ODNs containing these sequence motifs to be stronglyimmunostimulatory, resulting in the activation of B cells, NK cells, andantigen-presenting cells, and in the induction of a variety of cytokinesincluding interleukin-12 (IL-12), IL-6, and tumor necrosis factor-α. CpGODNs have also been found to be effective as adjuvants in inducingantigen-specific T-helper-1-like responses, and have been the focus ofmuch interest for their inclusion in anti-tumor vaccinations and use inother therapeutic applications (Klinman et al., 1999; Krieg et al,1999). Adjuvants enhance nonspecifically the immune response to anantigen. For example, pathogenic Arenaviruses appear to block or modifyimmunoregulatory cell signaling pathways (Peters & Zaki, 2002, Solomonand Vaughn, 2002; Fennewald et al., 2002). Using the present inventionit was possible to disrupt Arenavirus and Flavivirus cell signals thatcontribute to immune evasion and pathogenesis. Using thioaptamers it wasdemonstrated that the thio-modified aptamers of the present inventioncould be used to counteract viral induced cellular perturbations andprotect the infected host.

Viral Strategies to manage the host. During the co-evolution of virusesand their hosts, viruses have developed ingenious strategies tocounteract the host defenses that normally control viral replication andspread. Similarly, viral strategies modify the cellular environment topromote viral macromolecular synthesis and viral replication. Thishighly ordered interation often has the unfortunate consequence ofinducing disease in the host. Viruses have evolved mechanisms tointerfere with major histocompatibility complex antigen presentation,block apoptosis, disrupt complement cascades and modulate multiplecytokine networks (Lalani & McFadden, 1999; Ploegh, 1998). Viruses havetargeted cell-signaling pathways involved in cytokine and chemokinesignaling, the regulation of apoptosis, and the cell cycle. Studies haverevealed a number of instances of direct viral intervention in thereceptor and receptor proximal signaling, as well as direct interactionwith signaling kinase cascades and transcription factors (McFadden etal., 1998; Ploegh, 1998; Hiscott, 2001; Hiscott et al., 2001). Mostexamples have come from large DNA viruses with sufficient codingcapacity to encode viral homologs of cellular proteins. These virusesuse molecular mimicry to exploit the cellular environment to promoteviral replication and antagonize the immune response to sustain theirsurvival in an immunocompetent host (Cameron et al., 1999; Willer etal., 1999; Hiscott et al., 2001). Influencing key transcription factorsthat regulate pro or anti-inflammatory cytokines is an efficient meansby which viruses could cripple multiple immune responses (Powell et al.,1996; Tait et al., 2000). The strategies employed by the smaller, lessgenetically complex viruses are equally elegant, and often even more ofan enigma.

Pichinde infection of guinea pigs is particularly suited to studies onthe immunomodulation by virus infection. There are two virus variantswith minimal genomic differences but profoundly different effects on theanimal. Infection by the P2 variant of virus results in mild illnessfrom which the animal recovers. Infection by the P18 variant results indeath. These two virus variants were used to distinguish an effectiveimmune response against the P2 virus, from an ineffective responseagainst the P18 virus.

Using the aptamers of the present invention, the differential effect ofvirus infection was identified as including a profound effect on thetranscription factors NF-κB and RBP-Jκ. Data generated by the presentinventors (Fennewald et al., 2002) showed differential alterations inthe transcription factors NF-κB and RBP-Jκ in P2 and P18 virus-infectedguinea pig peritoneal macrophages. The P2 variant shows less NF-κBpresent and a higher mobility RBP-Jκ complex. This observation was usedin an animal model of arenavirus disease in which two virus variantsdifferentially affect target cell signaling pathways. NF-κB andAP-1(CREB) family members are key regulators of the immune response andtranscription factors involved interferon response to virus infectionall are differentially induced in pathogenic Pichinde infections. Usingthe aptamers of the present invention infected hosts virulence wasreduced by modulating virus induced alterations in cellular signaltransduction.

Many of the signaling pathways and transcription factors activatedduring immune system activation lead to the synthesis of theinflammatory cytokines. Certain pathways require the expression ofvarious cytokines. The effect of the virus variants (and polyI/C) on theinduction of cytokines was determined. FIG. 2 is a graph that shows thatpolyI/C is an effective inducer of the proinflammatory cytokine TNF-α.Infection with P2 and P18 also alter the expression of this and otherinflammatory cytokines. In particular, P2 and P18 induced equallycytokines such as IL-6; which are moderately different in theirinduction of TNF-α and substantially different in IL-12 induction (FIG.3). Thus, differences in signaling and inflammatory responses areassociated with immune activation by P2 virus and poor activation by theP18 virus. For example, IL-12 is especially important in directing theanti-viral immune response to the effective Th1 cytotoxic T cellresponse (Seow, 1998). In addition to supporting the association withthe immune response, this data can be used to direct the transcriptionfactors to target. For example, IL-6 induction is similar for both virusvariants.

To target transcription factors key in regulating TNFα and IL12 andother key mediators of the immune response two thioaptamers wereproduced, XBY-6 (SEQ ID NO.: 1) targeting NF-κB p50 homodimers andXBY-S2 targeting AP-1, both with six dithio residues. In FIG. 4, XBY-S2(SEQ ID NO.: 2) is demonstrated to bind specifically to AP-1 proteins inpre-B cell nuclear extracts (70Z/3) and to human recombinant c-junprotein dimers (AP-1). In FIG. 5, supershift analyses indicate thatXBY-S2 binds to several members of the AP-1 protein family includingJunD, CREB and possibly ATF2, and c-Jun. The XBY-6 thioaptamer bindsspecifically to the NF-κB p50 (or p105) homodimer (FIG. 6). Macrophagecultures were treated with XBY-S2 and XBY-6 and nuclear extracts wereproduced to assay the effects of these thioaptamers on the DNA bindingactivities of the transcription factors to which they are targeted. InFIG. 7, macrophage cultures were treated with liposomes, and liposomecontaining the indicated thioaptamers overnight and nuclear extractsproduced and assayed using the indicated oligonucleotides. The XBY-S2thioaptamer efficiently eliminated transcription factor binding to theAP-1 oligonucleotide. In contrast, treatment with XBY-6 resulted in anincrease in the NF-κB DNA binding activity.

In order to determine the consequence of the elimination of AP-1 DNAbinding activity by XBY-S2, stimulated macrophage cultures wereincubated with the thioaptamer with PolyI/C and measured the elaborationof TNFα and IL-6 into culture media. The expression of both TNFα andIL-6 are increased in response to polyI/C (FIGS. 8 and 9). Pretreatmentof cultures with XBY-S2 thioaptamer increases the amount of bothcytokines produced in response to poly I/C. These results indicate thatelimination of AP-1 from cells by the XBY-S2 decoy thioaptamer increasesthe production of cytokines.

It has been suggested that arenaviral and West Nile pathogenesis is theresult of viral perturbation of the immune response resulting in theinappropriate expression of cytokines. Therefore, modulation of cellsignaling by appropriate thioaptamers could reverse the inappropriategene expression and help to alleviate the symptoms and perhaps preventhost death. Guinea pigs were treated with the XBY-6 thioaptamertargeting NF-κB p50 homodimers at days 0, 1, and 2 day relative to timeof infection with a lethal dose of Pichinde virus. FIG. 10 demonstratesthat the thioaptamer prolongs the survival of Arenavirus infectedanimals. A thioaptamer of the same base content but scrambled insequence and containing CpG islands did not prolong survival (B92; FIG.10). Using the XBY-S2 thioaptamer, 50-80% protection of mice from alethal West Nile virus infection was demonstrated (Tables 5 and 6) aswell as prolongation of Pichinde virus survival similar to XBY-6 (datanot shown).

TABLE 5 Female 3-4 week-old NIH Swiss mice were given aptamers at oneday before and 90 minutes before administration of 10 LD₅₀ WN virusstrain USA99b by the ip route. Group # surviving [%] AST (days ± SD) PBSonly 0/5 [0]  7.2 ± 0.4 Liposomes only 0/5 [0]  8.0 ± 0.7 XBY-S2 4/5[80]  9 XBY-6 4/5 [80] 11

Based on the preliminary results obtained with XBY-6 thioaptamer andPichinde virus, it was determined if XBY-6 or XBY-S2 would have anyantiviral activity against flaviviruses. West Nile virus was selected asa model system due to its high virulence in the mouse model. Mice werechallenged with a low dose of virus (i.e., 30 pfu≈10 LD₅₀). Thethioaptamers (10 μg) were delivered IP in Tf×50 liposomes andadministered in two doses (one day before and 90 minutes before viruschallenge). Control mice given PBS or liposomes succumbed to WN virusinfection, while 80% of thioaptamer XBY-S2 treated animals survivedchallenge and remained healthy (Table 5). It was noted that boththioaptamers had antiviral activity. These results suggested that whilethe mechanism of protection may involve binding of XBY-6 to NF-κB orXBY-S2 to AP-1.

In previous studies with West Nile virus the present inventors hadobserved hat animals had a brief viremia that peaked on day 3 pi priorto viral brain invasion. As such, three animals from each test groupwere sacrificed on days 3 and 6 post infection to determine viremias andvirus infectivity levels in the brain. Accordingly, the protocol fromthe first study was repeated with increased group sizes of 16 mice (ofwhich 6 would be sampled) and increasing the virus challenge to 100 LD₅₀virus. As shown in Table 6, the initial results were reproducible. Bothcontrol groups (PBS and liposomes) succumbed to challenge with WN viruswhile the thioaptamer-treated mice survived and remained healthy. Theproportion of mice treated with XBY-S2 thioaptamer who survivedchallenge was the same in both studies (80%) while XBY-6 treatmentprotected 50% of mice in the second study as compared to 80% of mice inthe first study. These differences were not statistically significantgiven the small sample sizes.

To obtain fundamental information on the mechanism of protection,viremias and brain infectivity titers were measured in three micesampled from each group on days 3 and 6 post infection (Table 7). Asexpected, viremias and brain infectivity titers in the control (PBS andliposome) groups detected on day 3 prior to invasion of the brain andvirus detectable in the brains on day 6 post infection. The thioaptamertreated mice had reduced or undetectable viremias on day 3 postinfection and no detectable virus infectivity in brains on day 6 postinfection. These data indicate that the thioaptamer causes a reductionin the extraneuronal replication of the virus (as seen in the reducedviremias) and that there is insufficient virus to invade the centralnervous system and cause encephalitic disease. The difference betweenvirulent neuroinvasive strains of WN virus and poorly neuroinvasiveattenuated WN strains may be explained by these results. Two mechanismsseem possible, although the invention is in no way limited byhypothesis: 1) first, the thioaptamer induces an immune response againstWN virus; or 2) the thioaptamer blocks the WN virus replication. Thethioaptamer may be inducing localized interferon (or other mediators ofthe innate immune response) that inhibits replication of the virus sincethe thioaptamer includes double-stranded DNA while double-stranded RNAis known to be an efficient inducer of interferon.

TABLE 6 Study 2: Female 3-4 week-old NIH Swiss mice were given aptamersat one day before and 90 minutes before administration of 100 LD₅₀ WNvirus strain USA99b by the ip route. Group # surviving [%] AST(days ±SD) PBS only 0/10 [0]  8.3 ± 0.8 Liposomes only 0/10 [0]  7.7 ± 1.1XBY-S2 8/10 [80] 8.5 ± 0.7 XBY-6 5/10 [50] 8.0 ± 0.7

To investigate the activity of the modified thioaptamers and theantiviral mechanism of action of the thioaptamers, the susceptibility ofthioaptamer-protected mice virus to challenge was tested.Thioaptamer-treated mice from the second study who survived WN virusinfection were challenged at 21 days post-infection with 100LD₅₀ of WNvirus. All mice, including mock-infected controls from study 2 succumbedto virus challenge. This result indicates that there was insufficientvirus replication in thioaptamer-treated mice to induce an adaptiveimmune response. This would suggest that the mechanism of action of thethioaptamer is either innate immunity or direct antiviral activity ofthe thioaptamer.

Whether thioaptamers exhibited direct antiviral activity in cell culturewas also determined. The direct antiviral activity of the thioaptamerwas investigated in cell culture. Using six-well dishes containing Verocells, duplicate wells were treated with one of the

TABLE 7 Viremia and brain infectivity titers for Study 2 (see Table 6)Day 3 Day 6 Serum titer Brain titer Serum titer Brain titer Sample(pfu/mL) (pfu/brain) (pfu/mL) (pfu/brain) XBY-6 #1 30,000  —* — — XBY-6#2 — — — — XBY-6 #3 700 — — — XBY-S2 #1 100 — — — XBY-S2 #2 — — — —XBY-S2 #3 — — — — Lipo #1 2,000 — — 500,000 Lipo #2 2,500 — — 6,500,000Lipo #3 15,000 — — 3,500 PBS #1 25,000 — 100 5,500,000 PBS #2 20,000 — —180,000,000 PBS #3 4,500 — — 2,500,000 *indicates no virus detected;limits of detection were 50 pfu/ml of serum and 25 pfu/brain

1. Liposomes + xbyc2 (10 μg/well) 2. Liposomes + xbys1 (10 μg/well) 3.Liposomes + XBY-S2 (5 μg/well) 4. Liposomes + XBY-S2 (10 μg/well) 4.Liposomes only 5. Buffer only

Wells were incubated for 12 hours with the samples above and thenchallenged with WN virus at a multiplicity of infection (MOI) of 0.1.Samples were harvested from each well at 0, 14, 24, 34 and 48 hours. Nocytopathic effect was seen until 48 hours post virus infection. Eachwell was assayed at each time point by hemaggluttination (HA) assay todetect the presence of virus particles. All samples showed no detectableHA (i.e., ≦4 HAU) except for the samples at 48 hours post virusinfection when all wells had 32-64 HAUs. These results demonstrate thatthe thioaptamers have no direct antiviral activity.

One potential explanation for the antiviral activity of thioaptamers isinduction of interferon. This hypothesis was investigated by takinggroups of four 3-4 week-old female NIH Swiss and treat them with either10 ug of XBY-S2 in liposomes, liposomes only, or buffer only on day 0and day 1 post infection, followed by sacrificing mice on day 2 postinfection. Serum samples were diluted 1 in 3 and run in ELISAs to detectmouse interferon-α/β, interferon-γ, or TNF-α. None of these cytokineswas detected in the serum of any of the 12 mice sampled suggesting thatinterferon was not involved in the antiviral activity induced bythioaptamer XBY-S2.

FIG. 10 and Tables 5 and 6 demonstrate that the survival of P18 virusinfected animals can be prolonged using thioaptamers and thioaptamerscan protect the majority of the animals infected with West Nile virus.These results demonstrate that modified thioaptamers alter the outcomeof in vivo viral infections by Category A and B agents by themanipulation of transcription factors involved in the immune response.

FIG. 10 is a graph that shows survival curves following Pichinde P18infection in guinea pigs treated with the NF-κB aptamer, XBY-6, thescrambled control, B92, or vehicle, MT, of animals infected by injectionof 1000 pfu of Pichinde P18 at day 0, treatment consisted ofintraperitoneal injections at days 0, 1 and 2;

FIG. 11 is a graph that shows survival curves of guinea pigs withthioaptamers for infection by arenavirus. FIG. 12 is a graph that showssurvival curves following West Nile Virus infection in guinea pigstreated with the NF-κB aptamer XBY-6, the AP-1 aptamer XBY-S2, or theliposome vehicle of animals infected by injection with lethal doses ofWest Nile Virus.

SELDI MS Detection of NF-κB bound to Thioaptamer Surfaces and Beads. Thepresent inventors have demonstrated that thioaptamers bind bothpurified, recombinant NF-κB p50 and nuclear extracts on either beads (orCiphergen PBSII ProteinChip surfaces). FIGS. 13A-C are SELDI MS of p50binding to various ProteinChips and beads. In FIG. 13A, Ciphergen'sSELDI mass spectrometric methods were used to detect recombinant p50with using epoxy-activated ProteinChip Arrays. Duplex aptamers with a5′-amino terminus linked to a 12 carbon chain were synthesized. Theseduplex aptamers were the dithioate 14-mers XBY-6 (C12-XBY-6), the normalphosphate backbone 22-mer NF-κB binding site with the C12 5′-aminolinker (C12-IgκB) or a non-specific, non-covalently linked duplex(polydIdC) as a control. These aptamers were spotted individually ontospots of a preactivated ProteinChip Array (PS20) in 2 μl of 25 mM NaHCO₃(pH 9) and incubated overnight at room temperature and high humidity.Following incubation, excess aptamer was removed by washing 2 times in 5μl 25 mM PBS, 0.1% Triton X-100 (pH 7.2) and the surface was blocked tolimit non-specific binding with 1 μl of 100 μM bovine serum albumin for4 hrs. After blocking, excess BSA was washed away as above. Next, 4.3pmol recombinant p50 was spiked into 100 pmol BSA in 5 μl of optimizedEMSA buffer containing 20 mM DTT, 0.01 μM polydIdC and incubated on eachof the aptamer/thioaptamer surfaces for 2 hrs at room temperature andhigh humidity. Following incubation, each spot was washed with 5 μl of50 mM Tris buffer (pH 7.2), 0.1% CHAPS, 1 M urea, 0.5 M NaCl, followedby a water wash to remove all non-specific binding components. 0.8 μlSinapinic acid (saturated solution in 50% acetonitrile, 0.5%trifluoroacetic acid) was added to each spot, dried and the arrayanalyzed in the mass reader. As shown in FIG. 13, the p50 (MW ˜46,200)on either the XBY-6 or IgκB bound surfaces was detected, but not thecontrol. In other spectra with more stringent washing, the XBY-6 spot,but not the IgκB spot, was shown to retain the bound p50 (spectra notshown), confirming the tighter binding of p50 to XBY-6 (sub-nM) relativeto IgκB (K_(D)4 nM).

FIG. 14 shows that the XBY-6 thioaptamer can also capture recombinantp50 (MW ˜46,200) on gel beads to which the 5′ amino-C12 linked XBY-6 iscoupled to 20 ul (1:1) AminoLink® Plus Coupling gel (Pierce,Immunoprecipitation kit, cat # 45335). In this study, 3 μg of C12-XBY-6was coupled overnight at 4° C. following the kit protocol. Afterquenching the gel, 6 μg of p50 in 1 X EMSA buffer with polydIdC wasadded to the gel and incubated for 2 hrs with shaking at roomtemperature. The gel was washed to remove nonspecifically boundproteins, followed by one quick rinse with water. Protein bound to thegel was extracted with 5 μl of organic solvent (50% AcN and 0.01% TFA)with shaking for 20 min. All of the extracts were spotted onto NP20ProteinChips, dried, followed by addition of saturated SPA and read onthe Ciphergen PBSII MS (top two spectra). After extraction, 1 μl of thegel was loaded onto NP20 chip (bottom two spectra). Proteins still boundto the gel was analyzed using saturated SPA on the PBSII. Once again itwas found that p50 can be identified by SELDI, both in the extract andretained directly on the beads.

FIG. 15 shows the capture of nuclear extracts onto Ciphergen's PS20ProteinChip Arrays: Either 0.5 μg of C12-XBY-6, 0.25 pm of C12-IgκB or0.5 μg of poly dIdC were incubated on PS20 chip overnight. The chipswere blocked with 7 mg/ml BSA in PBS/0.1% Tween-20. Following blocking,49 μg of nuclear extract in optimized EMSA buffer were incubated on eachspot for 2 hr with shaking. Each spot was washed with PBS/0.1% Tritonthree times, followed by one quick wash with water. Proteins bound oneach spot were analyzed using saturated SPA on the PBSII. These resultsindicate that a protein was bound with a MW ˜105,591, which mayrepresent p105, the precursor to p50 or the p50/p50 homodimer.

Bead-based phosphorodithioate and phosphorothioate thioaptamercombinatorial libraries and high throughput sorting against targetedproteins. The one-bead, one-aptamer split synthesis method disclosedherein was used to identify a specific ODN aptamer that targets proteinsor other biomolecules. In combination with the split and pool synthesiscombinatorial chemistry method for creating a combinatorial library ofoligonucleotide agents (either phosphate, monothiophosphate ordithiophosphate; Gorenstein et al, U.S. Patent issued, 2002 and pending,1999a,b, 2001, 2002; Yang et al., 2002, relevant portions incorporatedherein by reference) both monothiophosphate and dithiophosphatecombinatorial libraries attached to individual support beads were shownto produce aptamers that demonstrate target-specific binding. Proteinsthat bind tightly to only a few of the 10⁴-10⁸ different support beadsmay be selected by binding either purified proteins, nuclear orcytoplasmic extracts or pools of proteins to the beads and thenidentifying which beads have bound target protein by immunostaining,fluorescent staining techniques or MS (SELDI). Thus, the methods andcompositions created and isolated thereby allow for rapid screening,isolation and identification of specific thioaptamers that bind toproteins such as NF-κB and AP-1 using the PCR-based identification tagof the selected bead disclosed herein.

Preparation and composition of the thioaptamer libraries and librariesof libraries. Depending on the nature of the targeted protein,thioaptamer combinatorial libraries were created that cover appropriatesequence space relative to the targeted protein. For transcriptionfactors duplex thioaptamers were create that have a significantpopulation of sequences similar to the consensus sequence. In the caseof the in vitro combinatorial selection approach disclosed herein, thecomplexity of the library can be as large as 10¹⁴ different sequencesand thus can cover all sequence space for a small (<22 nt) duplex. Forthe bead-based thioaptamer libraries, complexity is limited to thenumber of different beads—10⁶-10⁸, depending on their size.

To increase the complexity of the libraries one may also use a noveliterative approach in which a bead-based library of libraries ofthioaptamers is made in which as many as 10⁶ different thioaptamers areattached to a single bead and thus have a total complexity of as many as10¹²-10¹⁴ sequences in the library of library. For example, a library oflibraries was prepared on a 1 μmole scale of polystyrene beads (60-70μm). The downstream and upstream primers, 5′-d(GGATCCGGTGGTCTG)-3′ (SEQID NO.: 26) and 5′-d(CCTACTCGCGAATTC)-3′ (SEQ ID NO.: 27) weresynthesized in parallel on a two-column DNA synthesizer (Expedite 8909,Applied Biosystems). Following the 5′-primer, the sequences programmedon the synthesizer for the combinatorial library were5′-AT*GN*GA*AT*TT*NC*CA 3′ (SEQ ID NO.: 28) on column 1 and5′-GG*AG*NG*CN*CA*GG*AC-3′ (SEQ ID NO.: 29) on column 2. The 3′-primersequence completed the 44-mer programmed on the synthesizer. A “splitand pool” was used at each position indicated by an asterisk in order tosynthesize the combinatorial region for the library of libraries. Theletter N indicates a mixture of four bases (A, C, G and T). Five of thebeads were randomly selected from the library and “one bead one PCR” wasrun, cloned and sequenced. The results listed below indicated thesuccessful construction of the library of libraries.

E45-2-1: 5′-GG AG GA CT TT CC AC-3′ (SEQ ID NO.: 30) E45-2-2: 5′-GG AGGA CA TT GC AC-3′ (SEQ ID NO.: 31) E45-2-4: 5′-GG AG GA CC TT CC AC-3′(SEQ ID NO.: 32) E45-2-5: 5′-GG AG GA CC TT GC AC-3′ (SEQ ID NO.: 33)E45-2- 5′-GG AG GA CN TT TC AC-3′ (SEQ ID NO.: 34) 11: E45-2- 5′-GG AGGA CC TT TC AC-3′ (SEQ ID NO.: 35) 12: E45-3-1: 5′-GG GA TG GT CA GGAC-3′ (SEQ ID NO.: 36) E45-3-3: 5′-GG GC GG AT CA GG AC-3′ (SEQ ID NO.:37) E45-3-5: 5′-GG GA AG AT CA GG AC-3′ (SEQ ID NO.: 38) E45-3-6: 5′-GGGG TG AT CA GG AC-3′ (SEQ ID NO.: 39) E45-3- 5′-GG AG TG CT CA GG CA-3′(SEQ ID NO.: 40) 11: E45-6-1: 5′-GG AG CG GT GT CC AC-3′ (SEQ ID NO.:41) E45-6-2: 5′-GG GA GG GA TT AC CA-3′ (SEQ ID NO.: 42) E45-6-3: 5′-GGAG CG GT TT GC CA-3′ (SEQ ID NO.: 43) E45-6- 5′-GG AG CG AT TT CC CA-3′(SEQ ID NO.: 44) 10: E45-6- 5′-GG AG AG GT TT TC CA-3′ (SEQ ID NO.: 45)11: E45-7-1: 5′-AT AG GG CA CA GG AC-3′ (SEQ ID NO.: 46) E45-7-2: 5′-ATAG NG CC CA GG AC-3′ (SEQ ID NO.: 47) E45-7-5: 5′-AT AG GG CG CA GGAC-3′ (SEQ ID NO.: 48) E45-8-1: 5′-GG AG GG CC CA GC AC-3′ (SEQ ID NO.:49) E45-8-2: 5′-GG AG AG CA CA TC AC-3′ (SEQ ID NO.: 50) E45-8-3: 5′-GGAG CG CG CA CC AC-3′ (SEQ ID NO.: 51) E45-8-4: 5′-GG AG CG CG CA GCAC-3′ (SEQ ID NO.: 52) E45-8-5: 5′-GG AG GG CT CA GC AC-3′ (SEQ ID NO.:53) E45-8-6: 5′-GG AG AG CA CA AC AC-3′ (SEQ ID NO.: 54) E45-8- 5′-GG AGCG CG CA TC AC-3′ (SEQ ID NO.: 55) 10: E45-8- 5′-GG AG AG CG CA CC AC-3′(SEQ ID NO.: 56) 11:

For proteins in which there are no known sequence to design the library,the user of the present invention begins with a single-strand (ss) DNAor RNA thioaptamers with at least 30 nts in the randomized orcombinatorial regions. Using the methodology created and developed bythe present inventors for creating both duplex and ss DNA and RNAthioaptamer libraries by both enzymatic and bead-based methods. One suchtechnique is the one-bead, one-ODN library ligation reaction in whichshort (15 nucleotides) 5′- and 3′-sequences are sufficient to serve asprimers for bead-based PCR (Yang et al., 2002). To achieve even longercombinatorial segments, it is possible to eliminate entirely one of theprimer segments. High quality one-bead one-oligo libraries werecontracted by join two pieces of DNA based on an enzymatic ligationreaction or using highly active phosphorothioate towards 5′-iodo groupson the ODN. Standard phosphoramidite chemistry was used for synthesis of5′ monophosphate ODN (5′P(o)CCAGGAGATTCCAC-GGATCCGGTGGTCTGT-bead) (SEQID NO.: 57). The fully protected ODN with the non-cleavable linker beadswere treated with concentrated ammonia at 37° C. for 21 hours to removethe protecting groups while allowing the ODN to remain attached to thebeads. A selected single bead was mixed with the following components: 3μl of 40 μM 15 mer oligonucleotide (5′-CCTACTCGCGAATTC-3′, (SEQ ID NO.:27) 3 μl of 10× ligation buffer, 3 μl of DMSO, 2 μl of T4 RNA ligase and19 μl of ddH₂O. The reaction was performed at 5° C. for 17 hrs. Thesupernatant was removed carefully and washed with water. The single beadPCR reaction was run under established conditions. The PCR products wereanalyzed on a 15% native polyacrylamide gel. The PCR product was clonedusing the TA Cloning procedure (Invitrogen) and sequenced on an ABIPrism 310 Genetic Analyzer (Applied Biosystems). The desired sequence(5′-CCTACTCGCGAATTC-P(o)CCAGGAGATTCCAC-GGATCCGGTGGTCTGT-bead) (SEQ IDNO.: 58) was obtained.

These results show that the additional nucleic acid sequences may beadded to the one-bead, one-ODN library with high quality and efficiencywhile maintaining the integrity of the library. The ligation reactionallows longer random regions of aptamers to be synthesized on the beadswith higher yield since a primer region does not have to be stepwisesynthesized onto the bead sequence. The beads were screened for theability to bind the appropriate protein (such as the various NF-κBdimers or AP1 dimers) labeled with the Alexa Fluor 488 dye (MolecularProbes) or by binding fluorophor labeled antibodies as previouslydescribed. After thoroughly washing the protein-bound beads with PBS and0.1% Tween 20 to minimize nonspecific binding, the beads are sortedusing a multicolor flow cytometry and cell/bead sorting to visualize andsort the protein-bound thioaptamer beads and select the tightest bindingthioaptamer-protein complexes as shown in FIG. 6. The most intenselystained beads will be retrieved. Initially, the inventors concentratedon the NFκB and AP-1 dimers, but these methods may be applied by toother proteins involved in the immune response. Multicolor flowcytometry was capable of sorting at speeds of 10⁸ beads per hour orviewed in terms of assays for thioaptamers binding to target proteins,10⁸ assays per hour.

High throughput sorting (HTS) of homo- and heterodimers to thioaptamersby multi-color flow cytometry using multi-color flow cytometry HTS maybe used to select thioaptamers that bind preferentially to heterodimersof proteins. As described above, one monomer is tagged fluorescently (A)with a dye (cy3 for example) and a different monomer (B) with anotherdye (cy5 for example). Both proteins are mixed together and allowed tobind to the bead thioaptamer library. Next, two-color flow cytometry isused to compare cy3/cy5 color levels of each bead. To select homodimersthat have high affinity for homodimer A.A, beads that have high cy3levels and low cy5 levels are selected. Conversely, high cy5/low cy3indicates a thioaptamer sequence with selectivity for the B.B dimer. Forheterodimers, beads are selected for cy3/cy5 levels close to 1. SELDI MSmay be used to determine which proteins have been bound to selectedcombinatory thioaptamer beads and also used with single bead PCR toidentify which bead(s) in the combinatorial library have bound toprotein(s).

More than 2 dyes and multi-color flow cytometry may be used to selectvarious multimers. Thus, for NF-κB, at least 3 of the 5 differentmonomeric forms of the protein are combined, each with a differentfluorophor and use 3-color flow cytometry to select thioaptamers thathave high affinity and selectivity to homodimers A.A, B.B, C.C andvarious heterodimeric forms from the libraries. In principle, there arefew limits to the number of detectable markers (e.g., fluorochromes)that may be used with the present invention, e.g., 5-color flowcytometry may be used.

Sequencing may also be performed directly on the bead. Each individuallyselected bead is washed thoroughly with 8 M urea (pH 7.2) to remove theprotein and directly used for “one-bead one-PCR” amplification using the5′ and 3′ end primers (Yang, et al. 2002). The PCR products are TAcloned and sequenced as previously described to create hybridthioaptamers with normal phosphate, monothiophosphate, anddithiophosphate mixed backbones as well, keeping the total thiophosphatebackbone below 80% to minimize “non-specific” sticking.

The current approach demonstrated in the above examples requires adifferent nucleotide sequence to identify a backbone modification.Thioaptamer libraries were also created that only differ in the positionof phosphate or dithioate but not in its base sequence. It has beenshown that the positions of thiophosphates in a mixed backbone S-ODN canbe determined by reaction of the S-ODN with iodoethanol followed by basecatalyzed cleavage of the thiophosphate triester. This approach was usedto identifying the location of monothio- and dithiophosphate linkages,independent of base sequence.

Massively parallel, thioaptamer bead-based hts of the host and pathogenproteome may be used with the thioselection technology (both enzymatic[S]-ODN and synthetic [S]-ODN/[S₂]-ODN) to develop thioaptamerstargeting very important proteins (e.g., NF-κB and AP-1) to identifypromising therapeutic leads. Up to 1000's of different proteins in humanand pathogen proteomes by using a massively parallel, thioaptamerbead-based HTS of the proteomes with specialized high-throughputmulticolor flow cytometry/bead sorting in conjunction with SELDI™mass-spectrometric methods to identify potential new therapeutic targetsboth of proteins involved in the immune response to BT viruses as wellas viral proteins. Thioaptamers may be identified to inhibit thedifferentially expressed proteins in host-pathogen interactions as wellas underlying immune response processes and so amelioratecytopathological immune responses resulting in shock or to enhance“innate immunity” to help mount a more effective immune response.

Mass spectrometric protein detection technology can be used to identifybound proteins using HTS of thioaptamer beads. This approach hassignificant advantages, since MS is more sensitive than fluorescentimaging and will be very useful for low-abundance proteins. In addition,if more than one protein binds to a given thioaptamer bead, then it willbe possible to identify and quantify these proteins by SELDI. This isparticularly helpful for identifying non-covalent dimers such as NF-κBor AP-1 (there are 22 different monomeric forms of AP-1 and thus inprinciple 100's of different combinations of dimers possible).

Thioaptamer proteomic arrays were used to demonstrate the use ofProteinChip array technology (e.g., Ciphergen) for proteinidentification of modified thioaptamer beads or surfaces. SELDI MScombines the well-established principles of solid-phase extraction andtime-of-flight mass spectrometry in a process known as surface enhancedlaser desorption/ionization time-of-flight mass spectrometry.ProteinChip Arrays may be customized by covalently attaching affinityreagents such as the modified thioaptamers to the spot surface. If thebiological marker to be detected is known and thioaptamer affinityreagents are available, affinity surfaces can be designed to make use ofthis specific thioaptamer-protein interaction. Also, because SELDI usesmass spectrometric detection, several assays can be multiplexed easilyby taking advantage of the unique masses of each bound protein.

High-throughput screening (HTS) of thioaptamer libraries by flowcytometry and SELDI. Bead-based methods were used to identify boththioaptamer sequences and binding proteins in parallel, without the needto select one thioaptamer for each purified protein. A number of [S]-ODNor [S₂]-ODN combinatorial libraries are synthesized, each containing 10⁶to 10⁹ different, but related members (or a library of library with upto 10¹⁴ sequences). The solid-phase split synthesis described herein maybe used to create thioaptamer-bound bead libraries (one bead, onesequence or one library) as above. Each library can be sufficientlydifferent to provide high affinity and selectivity to a small number ofcellular proteins (such as AP-1 or NF-κB-type sequences). One or more ofthe thioaptamer library beads are incubated with cellular extracts,washed thoroughly to remove weakly bound proteins and the bound proteinsvisualized by direct fluorescent staining with cy3, cy5, SYPRO Ruby, orother newer dyes for high sensitivity (sub-nanogram). Fluorescentlystained beads can be sorted in the high-speed cell/bead sorter for thetop 10² or more beads which have the highest amount of bound protein.The beads selected with the greatest amount of protein bound will thenbe analyzed by SELDI MALDI-TOF mass spectrometric techniques determinewhich proteins are bound to each bead; even if more than one proteinbinds to the bead, the thioaptamer may be used to identify a selectgroup of proteins in cell extracts. The beads selected are then analyzedby SELDI methods to identify if a fairly limited number of differentproteins are bound to the specific bead. Alternatively, proteolysis ofthe proteins on the bead with trypsin and analysis of the peptidefragments by LC MS/MS QTOF2 can be used to identify the proteins on eachbead. After removal of protein from the beads by detergent and urea, thethioaptamer sequence on the bead can be determined by the PCR “one beadsequencing” method disclosed herein. Thus, a random library of “stickybeads” is selected and an extract containing the complete proteome toidentify both the thioaptamer sequence on the single beads and theprotein(s) bound.

HTS of combinatorial libraries to protein mixtures. Besides using cellextracts, known mixtures of hundreds of commercially available proteins(cytokines, transcription factors, etc.) may be applied to the mixtureof thioaptamer bead libraries. HTS cell/bead sorting is used followed byMS identification of bound proteins. This involves direct SELDIdetermination of the protein or peptide fragmentation methods followedby MS identification of bound proteins. A major advantage in usingthioaptamers rather than beads with proteins or monoclonal antibodiesattached to them is that proteolysis and MS peptide identification isnot complicated by proteolysis of bait proteins or Mab's. This approachcan be used in parallel with other commercially available antibodies forvirtually any protein (particularly AP-1), and serves as an alternativeto the more general screening of the complete proteome andidentification by SELDI MS methods alone. Once the sequences of thethioaptamers are identified, these are synthesized in larger quantitiesas reagents for diagnostics and therapeutics.

HTS of Thioaptamers Targeting Differentially Expressed Proteins in theProteome in virus infected cells. The thioaptamer-based multi-color flowcytometry HTS may also be used for targeting differentially expressedproteins within the host and pathogen proteomes, combined with MSdetection (SELDI). The thioaptamer bead-based combinatorial library canbe used in conjunction with fluorescent tagging of proteins followed bySELDI MS to identify proteins differentially expressed in control vs.virus infected cells. In this simple two-color assay, a combinatoriallibrary (or a combinatorial library of libraries) of thioaptamer beadsmay be synthesized, each bead with a single thioaptamer sequence (or acombinatorial library of thioaptamer sequences on each bead). Up to 10⁸beads can be created with a single thioaptamer sequence on each bead.Cell extracts of a sample such as uninfected cells is labeledfluorescently with a dye (cy3 for example) as carried out previously anda virus-infected cell extract is then labeled fluorescently with anotherdye (cy5 for example). Both cell extracts are mixed together and allowedto bind to the bead thioaptamer library. Next, two-color flow cytometryis used to compare cy3/cy5 color levels of each bead. If cy3/cy5 leveldiffers significantly (>2-fold) from 1, then the bead was captured. Todetermine which protein(s) have been bound to selected thioaptamer bead,SELDI MS will be used to characterize the bound target further. SELDI MScan be used to determine which proteins have been bound to selectedcombinatory thioaptamer libraries and also used with single bead PCR toidentify which bead(s) in the combinatorial library have bound toprotein(s). As shown above, Ciphergen's ProteinChip epoxy modifiedsurfaces may be used to covalently attach 5′-amino-linker thioaptamersto beads. Ciphergen's ProteinChip array technology allows forsolid-phase extraction to desorb more weakly bound proteins tothioaptamer surfaces, followed by surface enhanced laserdesorption/ionization time-of-flight mass spectrometry (SELDI-MS). Otherdiseases besides viral infections may be similarly targeted using thethioaptamers, systems and methods disclosed herein.

HTS of thioaptamers targeting differentially expressed proteins in theproteome in virus infected cells relative to treated cells (“HighThroughput Pharmacoproteomics”). In this embodiment, three-colorthioaptamer library bead sorting is used. In this three-color assay, acombinatorial library (or a combinatorial library of libraries) ofthioaptamer beads is synthesized, each bead with a single thioaptamersequence (or a combinatorial library of thioaptamer sequences on eachbead). Up to 10⁸ beads with a single thioaptamer sequence on each bead(or 10¹⁴ sequences on the library of libraries) are made. Uninfectedcell extracts (or control extracts) are labeled fluorescently with a cy3for example. A virus-infected cell extract (or any disease cell extractsuch as cancerous cells) is labeled fluorescently with cy5, and then athioaptamer therapeutic treated, virus infected (or other disease) cellculture is labeled with a third dye. The three proteome cell extractsare mixed together in equal total protein quantities and allowed to bindto the bead thioaptamer library (or library of libraries). Three-colorflow cytometry is used to compare cy3/cy5/dye 3 color levels of eachbead. If cy3/cy5 level differs from 1 (uninfected vs. infected) andcy5/Sypro Ruby differs from 1 (infected vs. infected and treated)differs from 1, then the bead can be captured. Such a control assuresthat the thioaptamer drug previously identified as a promising lead doesaffect specific protein levels. To determine which protein(s) have beenbound to selected thioaptamer beads, SELDI MS can be used tocharacterize the proteins bound to the target bead.

In one embodiment of the invention a complex of combinatorial librariesare created in which multiple transcription factor-like sequences withvarying thiophosphate substitution patterns are concatenated in a singlelong sequence so that it can bind to multiple transcription factors suchas NF-κB, AP-1, SP-1, GRE, SRE, etc., requiring a thioaptamer sequenceof at least 20-40-mers. These embodiments provide an attractive approachto defining therapeutic strategies in which multiple proteins can betargeted with multiple thioaptamers. Such a combination (adjuvant) ofdrug therapeutics is needed to improve immune responses in cancer, AIDS,etc. Mammalian protein signaling pathways are often redundant so that ifone pathway is affected, another can take over control. By perturbingmultiple, highly interwoven pathways, a greater opportunity to modulatethe immune response network is made available.

HT flow cytometry and bead selection. High-throughput screening (HTS) ofthioaptamer beads using high-speed multicolor flow cytometry/cellsorting is used. In principle, more than 10¹⁰ beads could be screenedwithin a single day, and specific bead subpopulations could be sortedfor subsequent proteomics analysis. This group also has considerableexperience in HTS of cells and bacteria (as well as beads) forsubsequent molecular characterizations by PCR and gene expressionmicroarray analysis.

Advanced HTS technologies may be used for large library screening andfunctional genomics. Single-cell (or bead) sorting of raresubpopulations may be used to isolate single beads from combinatoriallibraries. A special high speed sorter uses a unique two-stage signalprocessing system, configured in hardware as a single layer neuralnetwork, which allows for sophisticated cell or bead classificationsbased on multivariate statistics or learning through neural networks.

A 6-color high-speed flow cytometer/cell sorter is configured inhardware and software as a single-layer neural network that can also beused to generate real-time sort decisions on the basis of multivariatestatistical classification functions. While it can perform the usualtwo-way sorts it is commonly used in “straight-ahead” sorting mode toallow for extremely high sort recovery and purity at high throughputrates or to efficiently sort single cells for cloning or for subsequentmolecular characterizations by PCR.

Multi-color flow cytometry as a quantitation and validation tool forproteomics. These capabilities can also be used to sort for thioaptamersthat bind heterodimers or more complex protein mixtures. By usingdifferent fluorescently labeled dyes bound to specific proteins, beadsare sorted simultaneously that bind homodimers and heterodimers. Acovalently labeled p50 with Alexa-Fluor 488 dye was isolated (data notshown) and carried out 1- and 2-color thioaptamer bead sorting.

Production of large quantities of hybrid dithiophosphate aptamer. Usingchemistry developed independently in both Caruthers' and Gorenstein'slaboratory, the most promising dithioate hybrid backbone aptamers showgood in vitro and in vivo binding to the targets will be synthesized(Cho et al., 1993; Farschtschi & Gorenstein, 1988; Gorenstein et al.,1990; Gorenstein et al., 1992; Piotto et al., 1991) on a 5-10 μmolescale and purified (Mono Q; Yang et al., 1999; 2002).

Preparation of nuclear and cytoplasmic extracts was conducted at varioustimes after virus infection, and parallel uninfected control cultures of5×10⁷ cells are harvested and collected by centrifugation. Cell pelletsare resuspended and washed in phosphate buffered saline (PBS). Next,cells are lysed and the cytoplasmic and nuclear fractions isolated. Thenuclei are purified by centrifugation through a cushion of 2M sucrosebefore protein extraction. The protein content in all fractions will bedetermined by BCA Assay according the manufacturer's directions (Pierce,Rockford, Ill.).

Mass spectrometric identification of bound proteins. As demonstratedabove, sorted “positive” beads can be subjected to SELDI-MS analysis toconfirm the identity of the proteins bound to the thioaptamer beads ofthe present invention (via MALDI MS molecular ion characterization). Incases where the “positive” bead's thioaptamer might have bound not onlythe target protein but other proteins in a sample, e.g., a secondary oreven tertiary, etc. protein, SELDI-MS may be used to identify this eventthrough the detection of multiple molecular ions.

Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS). For proteinswhich cannot be identified from the MI, proteolysis and multidimensionalLC applying 2D chromatographic separation of peptides is used on-linewith MS analysis (Link et al., 1999; Washburn et al., 2001). This LCtandem MS approach is carried out using strong cation exchange (SCX)chromatography combined with reversed-phase (RP) chromatography. Using asalt step gradient, tryptic peptides of complexes are eluted from theSCX column onto the RP column, and contaminants of salts and buffers arewashed to waste using a diverter valve. Peptides are subsequently elutedfrom the RF column directly into the MS, either for mass fingerprinting,or for MS/MS sequence analysis. This LC tandem MS procedure is veryuseful for small amounts (femtomol) of complex. Yet another procedure istandem LC/tandem MS. The proteomes can be either human, GP, hamster ormouse-human and mouse genome databases are available.

LC or 2D SDS-PAGE and MS. These techniques are currently the majoranalytical tools used to identify proteins in the proteome. Thioaptamerbead libraries may be used to differentially screen the proteomes, using2D gel analysis for differential analysis of protein expression. Toimprove the comparative analysis of gel imaging software may be used toimprove result resolution, e.g., using Nonlinear USA, Inc. (Progenesis).The automated imaging features of this 2D imaging software reduce gelevaluation times substantially and are an important step towardshands-free analysis.

2D gel electrophoresis. 2D PAGE can be conducted essentially as firstdescribed by (O'Farrell, 1975). High-throughput may be employedPharmacia's IPGphor multiple sample IEF device or the first dimension,and Biorad's multiple gel SDS-PAGE systems (Protean Plus and Criteriondodeca cells) for the second. Gels will be stained with either SYPRORuby for high sensitivity (sub-nanogram) or Coomassie Blue when lesssensitivity is required. Image analysis of gels will be achieved with aPerkin Elmer (PE) ProEXPRESS Proteomic Imaging System using Nonlinear'sProgenesis imaging software. A Genomic Solutions' robotics recentlypurchased is utilized for protein spot picking and for sample trypsinhydrolysis (Proteomic Protein Picker), and sample clean-up, and sampleapplication to MALDI plates (ProPrep 4 Block System). Massfingerprinting for protein identification may use an Applied Biosystems(AB) matrix-assisted laser desorption/ionization (MALDI) time-of-flight(TOF) Voyager DE STR MS. Proteins will be identified with the Voyager'sProspector software. De novo sequencing and analysis ofposttranslational modifications can be achieved by electrospray (ESI)MS/MS (capillary LC nanoflow option).

Isotope-coded affinity tags (ICAT). Some differential protein expressionuse isotope-coded affinity tags (ICATs) for quantitative analysis ofcomplex protein mixtures (Gygi et al., 1999). In this procedure, thereis an option to fractionate proteins before to proteolysis decreases thecomplexity of proteins analyzed.

While this invention has been described in reference to illustrativeembodiments, this description is not intended to be construed in alimiting sense. Various modifications and combinations of theillustrative embodiments, as well as other embodiments of the invention,will be apparent to persons skilled in the art upon reference to thedescription. It is therefore intended that the appended claims encompassany such modifications or embodiments.

REFERENCE LIST

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1. A bead-based method for identifying both thio-modified backboneaptamer sequences and binding proteins comprising the steps of: A)incubating a partially thio-modified backbone aptamer bead library witha sample suspected of comprising one or more proteins, wherein each beadof the partially thio-modified backbone aptamer bead library comprisesmore than one copy of an unique aptamer having a unique sequence andbackbone modification; B) selecting one or more beads from thethio-modified backbone aptamer beads library to which the one or moreproteins are bound; C) identifying the one or more proteins by massspectrometry; D) identifying the unique sequence of the thio-modifiedaptamer; and E) locating the position of a backbone thio-modification ofthe unique aptamers comparing said sequence to positions encoded duringsplit synthesis.
 2. The method of claim 1, further comprising the stepof fragmenting the protein and separating the fragments by liquidchromatography followed by mass spectrometry after selection step B). 3.The method of claim 1, wherein the steps of identifying the protein bymass spectrometry, step C) is preceded by the steps of extracting andseparating the proteins by liquid chromatography.
 4. The method of claim1, wherein the steps of identifying the protein using mass spectrometrycomprises matrix assisted laser desorption ionization mass spectrometry.5. The method of claim 1, wherein the one or more thio-modified aptamersare attached to the beads by a non-cleavable linker.
 6. The method ofclaim 1, wherein the unique one or more thio-modified aptamers bound toone or more proteins are isolated from a gel.
 7. The method of claim 1,wherein the unique one or more thio-modified aptamers are attached tobeads and the one or more proteins that have bound the one or moreunique thio-modified aptamers are labeled and the beads are sorted basedon protein binding.
 8. The method of claim 1, wherein the one or moreunique thio-modified aptamers are attached to beads and the one or moreproteins that have bound the unique one or more thio-modified aptamersare labeled and the beads are sorted based on fluorescence.
 9. Themethod of claim 1, wherein the step of selecting one or more beads towhich one or more proteins has bound is defined further as beingfluorescent sorting using a flow-cytometer.
 10. The method of claim 1,wherein the one or more proteins is further defined as being obtainedfrom a cell extract.
 11. The method of claim 1, wherein the one or moreproteins is further defined as being obtained from a cell extract from avirally infected or diseased cell.
 12. The method of claim 1, whereinone or more thio-modified aptamers are attached to beads and the beadsare substantially protein-free.
 13. The method of claim 1, furthercomprising the step of sequencing the aptamer directly.
 14. The methodof claim 1, wherein the one or more beads comprises an [S]-ODN or[S₂]-ODN combinatorial library.
 15. The method of claim 1, wherein theone or more beads comprise a double-stranded thio-modified backboneaptamer library.
 16. The method of claim 1, wherein the librarycomprises sequence motifs for high affinity with cellular proteinsselected from the group consisting essentially of proteins that aremembers of the NF-kB protein family.